Pseudomonas 2009, XII International Conferencegenomics1.mh-hannover.de/pseudomonas2009/Startseite/Includes/img… · “Pseudomonas: Pathogenicity and ... Lecture Hall F August 13-17,

Pseudomonas 2009, XII International Conference

Dear friends and colleagues, dear guests,

as the president of the Hannover Medical School (MHH), I have the great pleasure and honor to cordially welcome you to the Pseudomonas Conference 2009 and I wish you an enjoyable, inspiring and constructive time on the campus.

Renowned speakers have agreed to share latest data and achievements with us at the Pseudomonas Conference this year and they will guarantee stimulating and informative discussions on Pseudomonas research. We are looking forward to a broad focus on basic microbiology and biotechnology research as well as on the clinical and practical relevance of the scientific findings. I would also like to stress the value of guest speakers and attendees for their contributions to this conference by submitting highly interesting abstracts.

The MHH is a medical faculty with university status, and a full-service health care institution. The clinic provides maximum medical care in 37 departments with more than 200.000 attended patients per year (50,000 inpatients and 150,000 outpatients). The MHH is internationally most renowned for transplant medicine and has a strong focus on infection, immunity and inflammation research. We have two pneumology departments dealing with cystic fibrosis (CF) of adults and children that provide care for about 500 individuals with CF and about 75% of all lung transplantations in Germany for patients with CF are performed at the Department of Heart Thorax and Transplantation Surgery at the campus. The presence of a clinical research group underscores the fact that CF research has a long tradition and is a strong focus at the MHH.

I am therefore looking very much forward to a stimulating and lively Pseudomonas conference 2009. I hope that you will enjoy your stay at the campus, meet friends and colleagues to exchange scientific ideas and initiate new collaborations and experience the hospitality of the town of Hannover.

Kind regards

Prof. Dr. Bitter- SuermannPresident, Medizinische Hochschule Hannover

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ORGANISERS

COMMITTEES

Scientific Advisory Committee

Pierre Cornelis, Bruxelles Alain Filloux, London Joanna Goldberg, Charlottesville Caroline Harwood, Seattle Roger Levesque, Quebec Victor de Lorenzo, Madrid Stephen Lory, Boston

Local Organising Committee:

Wolf-Rainer Abraham Susanne Häussler Dieter Jahn Dietmar Pieper Helga Riehn-Kopp Max Schobert Burkhard Tümmler Lutz Wiehlmann Elza Rakhimova Nina Cramer Colin Davenport Andreas Dötsch Sarah Frank Julia Garbe Marcela Heck Sonja Horatzek Macarena Marin Anya Pelnikevich Gesa Puls Florian Wölbeling

SPONSORS

Many thanks to Medizinische Hochschule HannoverUniversity of BrunswickHelmholtz- Zentrum für InfektionsforschungCentro Nacional de BiotecnologiaEmergenceTarpol KonsortiumMukoviszidose eV

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Contents

International Research Training Group (IRTG)

“Pseudomonas: Pathogenicity and Biotechnology” 6

Site Map 11

Programme 13

Abstracts 19

Keynote Lectures I 21

Genomics and Metagenomics 21

Metabolism and Physiology 32

Translational Research 47

Systems and Synthetic Biology 50

Ecology and Social Microbiology 54

Cell-to-Cell Communication and Intracellular Signalling 61

Pseudomonas as Pathogens 75

Cell Surface, Transport and Secretion 88

Keynote Lectures II 102

List of Authors 105

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The International Research Training Group (IRTG) „Pseudomonas: Pathogenicity and Biotechnology“

Host of the “Pseudomonas 2009” Conference is the International Research Training Group “Pseudomonas: Pathogenicity and Biotechnology”. This training program for graduate students has been running from the year 2000 until year 2009 and will terminate soon by September 2009. The Graduate School has been financed on the German side by the Deutsche Forschungsgemeinschaft for a period of nine years and from the Danish side over a period of five years. The partners are the Technical University of Denmark, The University of Copenhagen, the Technical University of Brunswick, the Helmholtz Institute for Infections Research in Brunswick and the Hanover Medical School.

The supervisors and the participating departments and institutions have a longstanding scientific interest in the Pseudomonas field and focus on fundamental and applied aspects of research on Pseudomonas aeruginosa and Pseudomonas putida: Genomics, microbial communities, biofilms and morphotypes, quorum sensing and signal transduction networks, cystic fibrosis, nosocomial infections, development of antipseudomonal agents, biocontrol and bioremediation.

During the nine-year running period the participating groups and organisations set up a mutually complementary and highly focused research program on Pseudomonas (see the table with the list of dissertations). The research activities were complementary. The groups located at the Technical University of Denmark put emphasis on projects dealing with biofilm production and signalling networks of P. aeruginosa and P. putida, while the groups in Brunswick focused more on the lifestyles of microbial communities, the construction of metabolic pathways and networks and on the biodegradation of toxic compounds in polluted soil and water by genetically engineered Pseudomonas strains. With respect to the medical impact of Pseudomonas, the German and Danish partners have a longstanding interest in the etiology, pathogenesis and epidemiology of the chronic airway infections with P. aeruginosa in individuals with cystic fibrosis. Major research topics are the defence mechanisms and immune response of the compromised CF host against P. aeruginosa and the genotypic and phenotypic adaptation of P. aeruginosa to the atypical niche of the CF lungs.

The teaching program concentrated on the training of the students in small groups, on joint platform activities of all participating institutions in terms of colloquia, workshops and summer schools. It is the policy of the IRTG to have a panel of supervisors with common scientific interest and diverse expertise to keep the number of projects small and to generate maximum synergisms and to recruit only the best students.

The Danish activities are summarised by the Danish speaker of the IRTG, Søren Molin:

“The Graduate School received funding for running expenses over a period of 5 years together with partial funding for a small number of student fellowships. Other graduate students working within the relevant topics were integrated in the Graduate School activities. Over the years we have together with our German partners organized biannual seminar series with mainly European scientists visiting in Hannover and in Lyngby. We have also in turns with the German partners organized Summer Schools. Finally, the Graduate School has catalyzed numerous joint projects between partners, both locally in Denmark and bilaterally with the German partners, especially in the areas of Pseudomonas pathogenicity and infections. Due to a change in the organization of Danish Graduate Schools 3 years ago the funding in Denmark for the European Graduate School stopped and the activities closed in 2008.”

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In summary, during the nine-year running period, the IRTG has been offering a highly streamlined and coherent research program on Pseudomonas pathogenicity and biotechnology with a diverse methodological spectrum and a broad study program in five centres of prime research on Pseudomonas as a model organism.

Burkhard TümmlerGerman Speaker of the IRTG

Helga Riehn-KoppCo-ordinator of the IRTG

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International Research Training Group (IRTG) „Pseudomonas: Pathogenicity and Biotechnology”:

Dissertations 2003 - 2009

Name, First NameTitle of Thesis

Allesen-Holm, MarieRole of extracellular DNA in Pseudomonas aeruginosa biofilm development

Bagge, NielsExpression of chromosomal beta-lactamase and transcriptome profile of biofilm growing Pseudomonas aeruginosa exposed to beta-lactam antibiotics

Bjarnsholt, ThomasEradication of chronic pulmonary infection caused by Pseudomonas aeruginosa in a murine model of cystic fibrosis, by interfering with the cell-cell communication

Bobadilla Fazzini, RobertoProteomics and kinetic modelling analysis of a 4-chlorosalicylate degrading bacterial community

Bohn, Tammy Yu-Sing, née ChangPseudomonas aeruginosa – mammalian host cell interaction: Phagocyte-sensitizing genes of P. aeruginosa

Calum, HenrikCharacterization of the murine innate immune response against planctonic and biofilm growing Pseudomonas aeruginosa infection in burns and lungs of mice

Cámara Herrera, Beatriz PatriciaEcological importance of a new metabolic route observed in Pseudomonas

sp. MT1 for the degradation of chlorosalicylates

Cramer, Nina, née WiwerinkMicroevolution of Pseudomonas aeruginosa in isolates from patients with cystic fibrosis

Gjermansen, MortenMicrobial biofilms: Programmed transition of bacteria from the sessile to the planktonic state

Hentzer, Patricia, née NikodemNew bacterial pathway of 4- and 5- chlorosalicylate degradation via 4-chlorocatechol and maleylacetate in a Pseudomonas strain

Hoffmann, NadineThe role of quorum sensing in the pathogenesis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis and antibacterial therapy

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Jordan, DorisCharacterisation of cytotoxic Pseudomonas aeruginosa “small colony variants” isolated from the respiratory tract from patients with cystic fibrosis

Kirkelund Hansen, SussePhysiological and genetic variation in mixed microbial surface communities

Klausen, MikkelInvolvement of bacterial migration in the development of multicellular structures in Pseudomonas aeruginosa biofilms

Klockgether, JensGene islands in Pseudomonas aeruginosa clone C.

Kumar Gopalaiah, VinodEuropean Cystic Fibrosis Twin and Sibling Study: Genetic Susceptibility to infectious disease

Lee, BaoleriCharacterization of genotypic and phenotypic diversity of Pseudomonas aeruginosa isolates during chronic lung infection in patients with cystic fibrosis

Mandsberg, LotteCharacterization of Pseudomonas aeruginosa mutators and DNA repair

Mortensen, NinellNanostructural investigation of pathogenic Gram-negative bacteria

Ostrup-Jensen, PeterCharacterization and modulation of the innate immune response during Pseudomonas aeruginosa lung infection in patients with cystic fibrosis: An experimental and clinical study

Pamp, Sünje JohannaDifferentiation in Pseudomonas aeruginosa biofilms

Pawelczyk, SonjaCarbon sharing of Pseudomonas spp in a 4- chlorosalicylate degrading consortium

Salunkhe, PrabhakarFunctional genomics of intracellular persistence of Pseudomonas aeruginosa in PMNs

Saravanamuthu, Senthil SelvanDifferential adaptation of Pseudomonas aeruginosa morphotypes to ion limiting conditions

Scheithauer, BrittaCharacterization of microbial biofilm communities occluding biliary stents

Schreiber, KerstinCharacterization of the Pseudomonas aeruginosa Usp-type stress protein PA3309

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Skindersø, Mette ElenaEurkariotic-prokariotic interaction during the infection process

Sriramulu, Diraviam DineshAdaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Wu, HongInvestigation of N-acyl homoserine lactone mediated quorum-sensing systems in the pathogenesis of chronic Pseudomonas aeruginosa lung infection

Würdemann, DiecoMobile gene islands of Pseudomonas aeruginosa

Ziegler, IsabelVirulence analysis of different morphotypes of Pseudomonas aeruginosa and of Burkholderia species

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HANNOVER MEDICAL SCHOOL

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POSTER SESSIONS (J2)

LECTURE HALL F (J1)

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Programme

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Pseudomonas 2009, XII International ConferenceMedizinische Hochschule Hannover, Lecture Hall FAugust 13-17, Hannover, Germany

Thursday, Aug. 13th

13.00 Registration opens

16.30-16.40 Welcome (Tümmler, B)

16.40-18.20 Keynote Lectures I (Timmis, K)

16.40 de Lorenzo, V; Madrid, Spain Engineering vs. tinkering the regulatory network of the TOL plasmid of Pseudomonas putida mt-2

17.30 Lory, S; Boston, USAPost-transcriptional regulation of Pseudomonas aeruginosa virulence gene expression

18.30 Get Together

Friday, Aug. 14th

09.00-10.30 Genomics and Metagenomics (Lory, S / Levesque, R)

09.00 Ussery, DW; Lyngby, Denmark Comparative Pan-Genomics of Pseudomonas

09.20 Klockgether, J; Hannover, Germany Genomic comparisons of Pseudomonas aeruginosa strains

09.40 Surette, MG; Calgary, Canada Synergistic Infections of Pseudomonas aeruginosa and Oropharyngeal Flora in a Drosophila Model of Polymicrobial Infections

10.00 Harmer, CJ; Sydney, Australia Genetic basis of increased virulence in frequent Pseudomonas aeruginosa clonal complexes isolated from Australian cystic fibrosis patients.

10.15 Kivisaar, M; Tartu, Estonia Evolution of bacterial genomes under environmental stress

10.30-11.00 Coffee Break

11.00-12.35 Metabolism and Physiology (Cornelis, P / Schobert, M)

11.00 Dietrich, LEP; Cambridge, USA Phenazines control gene expression and community behavior in Pseudomonas aeruginosa PA14

11.20 Blankenfeldt, W; Dortmund, Germany Kissing Butterflies and French Fries: The Biosynthesis of Pyocyanin in Pseudomonas aeruginosa

11.35 Reimmann, C; Lausanne, Switzerland A small RNA of Pseudomonas aeruginosa stimulates the

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expression of the pyochelin regulatory gene pchR

11.50 Frangipani, E; Lausanne, Switzerland Regulation of aerobic respiration in Pseudomonas aeruginosa PAO1, evidence for a novel regulatory protein

12.05 Bordi, C; Marseille, France Fine-tuned regulation of rsmY via the HptB phosphorelay pathway in Pseudomonas aeruginosa

12.20 Pflüger-Grau, K; Madrid, Spain Pyruvate dehydrogenase is the metabolic target of the EIIANtr component of the N-related phosphotransferase system (PTSNtr) of Pseudomonas putida

12.35-14.00 Lunch

14.00-15.25 Translational research (Wiehlmann, L / Pieper, D)

14.00 Ferrer, M; Madrid, Spain New insights into the Pseudomonas putida KT2440 reactome

14.20 Givskov, M; Copenhagen, Denmark Chemical biology, biofilms and PMN-Leukocytes

14.40 Flegler, KA; Essen, Germany Inhalation of acid sphingomyelinase inhibitors normalizes ceramide, inflammation and Pseudomonas aeruginosa infections in cystic fibrosis

14.55 Gross, H; Bonn, Germany Genomic Mining for Bioactive Small Molecules in the Genome of Pseudomonas fluorescens Pf-5 and P. syringae pv. syringae B728a

15.10 Ruijssenaars, H; Delft, The Netherlands Green production of substituted aromatics by engineered solvent-tolerant Pseudomonas putida S12


16.00-18.30 Poster session

Saturday, Aug. 15th

09.00-10.25 Systems and Synthetic Biology (Jahn, D / de Lorenzo, V)

09.00 Martins dos Santos, V; Braunschweig, Germany Metabolic comparison of the in silico phenotype-genotype relationship of Pseudomonas putida and Pseudomonas aeruginosa

09.20 Thiele, I; Iceland / San Diego, USASystems Modeling of Pseudomonadales : Biotechnological and Biomedical Applications

09.40 Papin, JA; Charlottesville, USA Integrating genome-scale transcriptional regulation and metabolism in Pseudomonas aeruginosa elucidates metabolic phenotypes relevant to its virulence

09.55 Borgos, SEF; Trondheim, Norway

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SCaRAB – Systems biology of Carbon Redirection in Alginate Biosynthesis

10.10 Martínez-García, E; Madrid, Spain Synthetic genetic tools for post-genomic analysis and engineering of Pseudomonas


11.00-12.30 Ecology and Social Microbiology (Abraham, WR / Molin, S)

11.00 Molin, S; Lyngby, Denmark Pseudomonas aeruginosa populations in airways of cystic fibrosis patients

11.20 Bruce, KD; London, UK Understanding lung disease in cystic fibrosis patients; the dynamics of Pseudomonas aeruginosa during infection

11.40 Buckling, A; Oxford, UK Social interactions and virulence of Pseudomonads

12.00 Schuster, M; Corvallis, USA Conflict resolution in bacterial quorum sensing by re-evolved cooperation

12.15 Barkovits, K; Bochum, Germany Pseudomonas aeruginosa and its phytochrome operon: More than a photoreceptor?

12.30-14.00 Lunch

14.00-15.40 Cell-to Cell Communication and Intracellular Signalling (Häussler, S / Givskov, M)

14.00 Parsek, MR; Seattle, USA The RSCV phenotype of Pseudomonas aeruginosa

14.20 Tolker-Nielsen, T; Copenhagen, Denmark Characterization of starvation-induced dispersion in Pseudomonas putida biofilms.

14.40 Navazo, A; Madrid, Spain Three independent signalling pathways repress motility in Pseudomonas fluorescens F113.

14.55 Fernández-Piñar, R; Granada, Spain Cell density dependent regulation in Pseudomonas putida KT2440

15.10 Lapouge, K; Lausanne, Switzerland How do RsmA/CsrA-like proteins specifically recognise their mRNA targets in Pseudomonas fluorescens CHA0?

15.25 Messina, M; Nottingham, UK Gene regulation in Pseudomonas : from environmental signals to responses via global post-transcription control and intracellular messaging


16.00-18.30 Poster session

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Sunday, Aug. 16th

08.30-10.10 Pseudomonas as Pathogens I (Döring, G / Goldberg, J)

08.30 Döring, G; Tuebingen, Germany Post-translational regulation of alginate export in Pseudomonas aeruginosa by a novel oxygen sensor

08.50 Hunter, RC; Cambridge, USA Identification of Factors Involved in Melanin Overproduction within the Cystic Fibrosis Lung

09.05 Storey, DG; Calgary, Canada Phenotypic diversity and interactions between Pseudomonas aeruginosa CF isolates from individual sputum samples

09.20 Malone, J; Basel, Switzerland A role for cyclic-di-GMP in persistent Pseudomonas aeruginosa infections

09.35 Fukushima, J; Akita, Japan The pathogenicity and regulated production of the membrane vesicle from Pseudomonas aeruginosa

09.50 Murphy, TF; Buffalo, USA The Many Faces of Pseudomonas aeruginosa in Chronic Obstructive Pulmonary Disease


10.40-12.00 Pseudomonas as Pathogens II (Döring, G / Goldberg, J)

10.40 Welch, M; Cambridge, UK Virulence factors associated with the sessile lifestyle.

10.55 Dudler, R; Zurich, Switzerland A virulence factor of the plant pathogen Pseudomonas syringae pv. syringae is a proteasome inhibitor

11.15 Lee, JH; Busan, South Korea Snatch of the host immunity by quorum-dependently secreted factors from Pseudomonas aeruginosa

11.30 Mooij, MJ; Cork, Ireland Pseudomonas aeruginosa pathogenesis in zebrafish embryos

11.45 Vallet-Gely, I; Gif sur Yvette, France Characterization of a new secondary metabolite that plays a key role in Pseudomonas entomophila virulence towards Drosophila melanogaster

12.00-13.00 Lunch

13.00-22.00 Social Event (Excursion to Hamelin with dinner at Hülsede castle)

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Monday, Aug. 17th

10.00-11.20 Cell Surface, Transport and Secretion I (Filloux, A / Tolker - Nielsen, T)

10.00 Burrows, LL; Hamilton, Canada Assembly of type IV pili in Pseudomonas aeruginosa

10.20 Ruer, S; Marseille, France The "P-usher", a novel protein transporter involved in fimbrial assembly and TpsA secretion

10.40 Mougous, JD; Seattle, USA Posttranslational regulation of type VI secretion in Pseudomonas aeruginosa

11.00 Attrée, I; Grenoble, France Biogenesis of the type III secretion system in Pseudomonas aeruginosa


11.45-13.00 Cell Surface, Transport and Secretion II (Filloux, A / Tolker - Nielsen, T)

11.45 van den Berg, B; Worcester, USA Structural and biochemical characterization of the OprD outer membrane channel family

12.00 Journet, L; Illkirch, France The fate of ferric-pyoverdin after uptake across the outer membrane of Pseudomonas aeruginosa

12.15 Schell, S; Jülich, Germany Molecular interactions of passenger and transport domains demonstrated with autotransporter chimera of Pseudomonas aeruginosa EstA and Pseudomonas putida EstP

12.30 Wood, LF; Richmond, USA Characterization of the Cell Wall Stress Response and Recovery through σ22 and its Controlled Factors in Pseudomonas aeruginosa

12.45 Rehm, BHA; Palmerston North, New Zealand Alginate polymerisation/secretion in Pseudomonas aeruginosa

13.00-14.15 Lunch

14.15-15.45 Keynote Lectures II (Tümmler, B / Wiehlmann, L)

14.15 Goldberg, JB; Charlottesville, USA The challenges of Pseudomonas vaccine development: from here to immunity

15.00 Hancock, R; Vancouver, Canada Antibiotic resistance, epithelial cell interactions and swarming motility in Pseudomonas as complex adaptations

15.45-16.00 Closing Remarks (Tümmler, B)

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Abstracts

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Keynote Lectures I

K-01 Topic: Systems, Synthetic and Computational Biology

Engineering vs. tinkering the regulatory network of the TOL plasmid of Pseudomonas putida mt-2Victor de Lorenzo1

1 Centro Nacional de Biotecnología, Madrid, Spain

P. putida mt-2 harbours plasmid pWW0 with two operons for metabolization of toluene and m-xylene. The upper operon encodes conversion of m-xylene into 3-methylbenzoate (3MB), while the meta operon determines transformation of 3MB into TCA substrates. The upper operon is expressed from the Pu promoter in the presence of the activator XylR and m-xylene, while promoter Pm drives the meta operon in response to the XylS activator and 3MB. The XylR protein also represses its own production and activates xylS expression. In order to picture the complete control circuit, we have deconstructed the extant TOL regulatory network into a whole of logic gates [1]. These represent both transcriptional and metabolic actions as a coherent set of binary responses (i.e., a logicome). Formalization of the interplay between regulatory components as Boolean operations has allowed the setup of a model that describes the inner logic of the regulatory architecture. To this end have used piecewise-linear (PL) differential equations for describing circuit dynamics in a numerical fashion. Simulations of the behaviour of the system were then performed using the Genetic Network Analyzer program (VisualGNA). By setting the system as cells in stationary phase and having m-xylene as the one external input, the model recreated faithfully the regulatory features described for the TOL circuit. The one salient element of the system was the redundant mechanism of Pm promoter regulation either by XylS hyperexpression or by co-activation with 3MB. The results of simulations that perturb this regulatory node suggest that Pm up-regulation through XylS without the concourse of an effector is necessary for programming cells for a rapid consumption of 3MB. This reflects the metabolic memory of the system for removal of 3MB before the product is actually formed as an intermediate of m-xylene degradation. Such a dual mechanism of Pm activation thus ensures that 3MB is channelled only through the TOL enzymes and it does not does not cause toxicity through its misrouting into the chromosomally-encoded benzoate degradation pathway [2,3]. Comparison of the TOL logicome with related catabolic pathways support the notion that metabolic traffic imposes distinct constrains on the architecture of the cognate regulatory circuits.[1] Silva-Rocha & de Lorenzo (2008) FEBS Lett 582: 1237-1244.[2] Dominguez-Cuevas et al. (2006) JBC 281: 11981-11991.[3] Velazquez et al (2005) Mol Microbiol 57:1557-1569.

K-02 Topic: Pseudomonads as Pathogens

Post-transcriptional regulation of Pseudomonas aeruginosa virulence gene expressionStephen Lory1

1 Harvard Medical School, Boston, USA

Bacteria that are common inhabitants of soil or water reservoirs cause some of the most serious infections of humans. Moreover, the ability of these opportunistic pathogens to thrive in a wide range of environments often depends on the activities of specialized signal transduction pathways and complex regulatory networks. Pseudomonas aeruginosa can cause serious acute infections in immunocompromized individuals, or chronic infections in patients with cystic fibrosis. We have recently uncovered a regulatory network that functions as a molecular switch controlling the expression of hundreds of genes, including those encoding acute toxic proteins, and in a reciprocal mode, the formation of biofilm determinants important for chronic infections. The switch operates by controlling the reversible phosphorylation of GacS/GacA two component system, which regulates transcription of only two genes, the small RNAs (sRNA) rsmZ and rsmY. The global impact of the GacS/GacA system is therefore due to post-transcriptional activities of these sRNAs, acting by antagonizing the binding of the translational regulator RsmA to its target sites at the 5` ends of transcripts. Another component of the regulatory switch is a signal transduction pathway utilizing the second messenger cyclic-di-GMP (c-di-GMP). This regulatory nucleotide also participates at post-transcriptional level in the reciprocal regulation of acute and chronic factors produced by P. aeruginosa. The interplay between the RsmZ/Y/A regulatory pathway and c-di-GMP activity will be discussed in the context of environmental adaptation of P. aeruginosa to human hosts in different infection settings.

Genomics and Metagenomics

Oral presentations

O-01 Topic: Genomics and Metagenomics

Comparative Pan-Genomics of PseudomonasDavid W. Ussery1

1 Center for Biological Sequence Analysis, DTU, Lyngby, Denmark

Currently there are about three dozen Pseudomonas genomes sequenced and available for analysis. The relationship between the pan- and core-genomes of four different Pseudomonas species will be examined: P. aeruginosa, P. fluorescens, P. putida, and P. syringae.

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Genomic comparisons of Pseudomonas aeruginosa strainsJens Klockgether1

1 Hannover Medical School, Germany

Pseudomonas aeruginosa isolates from different sources show a broad variety of phenotypic differences. These phenotypic variations are accompanied by genomic differences which are caused both by a highly variable accessory genome and differences in the species-specific core genome. We report on several studies analysing intra- and interclonal variations in P. aeruginosa with different approaches.Based on SNP-genotyping methods, a large collection of P. aeruginosa isolates from various habitats was screened to gain insight into the general population structure. Thus, a few globally distributed dominant clonal lineages were identified, but within these lineages various subsets of strains appeared in different environmental and clinical habitats.Next-generation sequencing has been applied to three different isolates each from selected clonal lineages. This approach allowed monitoring of intraclonal variations that occurred either in clonal variants colonising different environments or during persistence in the same habitat. In addition to this, selected pairs of sequential isolates from cystic fibrosis patients were compared by genome-wide hybridisation and sequencing methods. The respective strains were analysed to gain knowledge about microevolution of strains during adaptation to a particular habitat and its consequences.Microevolution must have occurred in the P. aeruginosa reference isolate PAO1 as well during recent decades, since experimental results for PAO1 may vary significantly, depending on the respective source of the used strain. Also genomic variations had already been indicated by pulsed-field gel electrophoresis. (Re-)Sequencing of two of the most relevant PAO1-sublines revealed a number of genomic differences in comparison to the commonly used reference sequence published in 2000, which have likely caused phenotypic variation of PAO1-sublines maintained in different laboratories.


Synergistic Infections of P. aeruginosa and Oropharyngeal Flora in a Drosophila Model of Polymicrobial InfectionsMichael G Surette1

1 University of Calgary, Canada

A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Cystic Fibrosis (CF) is a disease in which complex dynamic microbial communities colonize the airways of patients, however the clinical relevance of many of the organisms present is not known. Most are members of the oropharyngeal flora and are usually considered avirulent and not clinically significant. We have developed a novel Drosophila melanogaster infection model to study microbe-microbe interactions and polymicrobe-host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe-microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The results highlight the potential complexity of polymicrobial infections. The Drosophila model of polymicrobial infections not only allows relevant microbe-microbe interaction to be easily discerned based on fly survival but also provides a framework to further discriminate these interactions by assaying both bacterial and host gene expression in vivo. The polymicrobial infection model was used to differentiate the bacterial flora in CF sputum, revealing that a large proportion of the organisms in CF airways have the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa. These results suggest a role for organisms other than conventional pathogens in disease progression in CF and furthermore may provide alternative bacterial targets in the treatment of CF airway infections.

Poster/Oral presentations

PO-1 Topic: Genomics and Metagenomics

Genetic basis of increased virulence in frequent P. aeruginosa clonal complexes isolated from Australian cystic fibrosis patients.Christopher J Harmer1, Khaled Al Nassafi1, Jim Manos1, Honghuah Hu1, Barbara Rose1 and Colin Harbour1

1 Department of Infectious Diseases and Immunology, University of Sydney, NSW, Australia, 2006

BackgroundPreliminary studies genotyping P. aeruginosa isolates from cystic fibrosis (CF) populations indicate the epidemic spread of frequent clonal strains or complexes of P. aeruginosa within and between CF clinics in eastern Australia. Our recent data provide evidence of increased virulence of two frequent clonal complexes (FCC) of P. aeruginosa infecting approximately half of the adult CF population in a CF centre in eastern Australia. CF patients infected with FCC recorded more exacerbations and hospitalizations than those infected with infrequent clonal complexes (IFCC). This study seeks to establish whether the increased virulence observed clinically has a genetic basis.Objectives1. To compare the virulence of FCC and infrequent clonal complexes (IFCC) in the C. elegans virulence model.2. To determine the genetic basis of increased virulence using a broad array to identify novel strain-specific virulence genes in FCC and IFCC strains.MaterialsIn order to establish the genetic basis of virulence we sequenced the most dominant FCC in Sydney and Melbourne (AES-1R) and used this sequence, together with that of 7 other P. aeruginosa strains, to design a non-redundant array (PANarray) in collaboration with the

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Victorian Bioinformatics Consortium (VBC). Ethical approval and patient permission was obtained for these studies. The C. elegans was a sterile strain suitable for slow killing experiments and was obtained from the C. elegans Genetics Centre, USA.ResultsIn the C. elegans virulence assay (n=50), the average LD50 was 72 h for FCC (n=11) and 80 h for IFCC (n=11). The distribution of LD50

ranged between 50 h and 150h for FCC and between 36 h and 145 h for IFCC. There were virulent clones with short LD50 in both FCC and IFCC. Genomic DNA from clones showing highest and lowest LD50 is presently being compared using the PANarray, with the possibility that more virulent strains will be found to possess a common cohort of genes.ConclusionThe identification of virulent isolates of P. aeruginosa FCC using the in vivo C. elegans model is an important first step in the process of understanding virulence in CF strains. The further identification of conserved genes in virulent strains will allow us to undertake mutation studies that will help identify the essential virulence factors in these CF strains.

PO-2 Topic: Genomics and Metagenomics

Evolution of bacterial genome under environmental stressMaia Kivisaar1, Kairi Tarassova1, Mariliis Tark1, Signe Saumaa1, Riho Teras1, Andres Tover1 and Radi Tegova1

1 Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia

Bacteria can rapidly evolve under adverse environmental conditions. The diversity can arise both due to point mutations and various recombination events. Point mutations originate from various sources, including errors made during replication, mutagenic nucleotide substrates, and endogenous DNA lesions (e.g., oxidative damage). Bacteria have several specialized DNA polymerases which can bypass a variety of DNA lesions blocking DNA synthesis by a replicative DNA polymerase Pol III. Participation of specialized DNA polymerases in DNA repair synthesis has been suggested as well. We have previously shown that the generation of point mutations in carbon-starved P. putida is facilitated due to error-prone DNA synthesis by the specialized DNA polymerases Pol IV and the plasmidial Pol V homologue RulAB (1, 2). Additionally, DnaE2, which is a homologue of the alpha subunit of Pol III, and ImuB, related to Pol IV, affect occurrence of mutations in P. putida whereas DnaE2 reduces and ImuB elevates the mutation frequency (3). The results of our current studies provide experimental evidence for participation of specialized DNA polymerases in DNA repair synthesis in stressed cells. For example, Pol IV is recruited to the DNA replication machinery preferentially in DNA repair synthesis initiated by nucleotide excision repair (NER) and mismatch repair (MMR) enzymes (4). Moreover, DnaE2 and ImuB can partially complement the absence of Pol I in NER and perhaps also in other processes where Pol I is involved. Additionally, we have also studied the role of accumulation of reactive oxygen species (ROS) on mutagenesis in starving bacteria. The mutation frequency is specifically elevated in a starving RpoS-deficient P. putida which is more vulnerable to ROS than the wild-type strain. Also, the frequency of homologous recombination is drastically increased under the conditions of prolonged starvation of P. putida and therein the accumulation of ROS seems to play an important role as well. (1) Tegova, R., Tover, A., Tarassova, K., Tark, M., and Kivisaar, M. (2004) J Bacteriol 186: 2735-2744.(2) Tark, M., A. Tover, K. Tarassova, R. Tegova, G. Kivi, R. Horak, and Kivisaar M. (2005) J Bacteriol 187: 5203-5213. (3) Koorits, L., Tegova, R., Tark, M., Tarassova, K., Tover, A., and Kivisaar M. (2007) DNA Repair 6: 863-868.(4) Tark, M., Tover, A., Koorits, L., Tegova, R., and Kivisaar, M. (2008) DNA Repair 7: 20-30.

P-3 Topic: Genomics and Metagenomics

Screening of a Pseudomonas aeruginosa mutant library for phenotypes in antibiotic sensitivityCarolina Alvarez-Ortega1, Jorge Olivares P1, Alicia Fajardo1 and José Luis Martínez1

1 CNB-CSIC, Madrid, Spain

Pseudomonas aeruginosa exhibits a high intrinsic resistance to antibiotic therapy mainly due to low outer membrane permeability and to the presence of multiple efflux pump systems. In addition, P. aeruginosa develops increased resistance as a consequence of mutations and acquisition of resistance genes through horizontal gene transfer. As a result infections caused by this important opportunistic pathogen are very difficult to eradicate. We screened a comprehensive P. aeruginosa PA14 mutant library [1] for increased and decreased susceptibility to six different antibiotics in order to identify additional genes that contribute to intrinsic antibiotic resistance and also to gain a better understanding of the impact that some mutations might have on the P. aeruginosa response to antimicrobial therapy. The following antibiotics were used in the screening: polymyxin B, amikacin, imipenem, tetracycline, ceftazidime and ciprofloxacin. A total of 300 mutants showed an increase or a decrease in susceptibility to at least one out of the six antibiotics that we utilized. Among these we found mutants in genes involved in energy metabolism, flagellar biosynthesis and alginate production. In agreement with the high tolerance to antibiotic therapy that characterizes P. aeruginosa, we observed that the majority of the mutants found in our screening exhibited an increase in susceptibility to at least one antibiotic. A similar study with ciprofloxacin has been published [2] and the coincidences between the two studies validate our screening. 1. Liberati, N. T., J. M. Urbach, et al. (2006). An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. PNAS 103(8): 2833-2838.2. Breidenstein, E. B., B. K. Khaira, et al. (2008). Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility. Antimicrob Agents Chemother 52(12): 4486-91.


Genetic adaptation of Pseudomonas aeruginosa for the commitment to chronic lung infectionIrene Bianconi1, Andrea Milani2, Moira Paroni1, Roger Levesque3, Giovanni Bertoni2 and Alessandra Bragonzi1

1 San Raffaele Scientific Institute, Milano, Italy, 2 Università degli Studi di Milano, Milano, Italy, 3 Universitè Laval, Quebec, Canada

P. aeruginosa clones isolated from cystic fibrosis (CF) patients are capable of becoming more pathogenic if the functional modification or elimination of an original gene(s) changes their phenotype appropriately. This change-of-function mechanism depends on the occurrence of genetic mutations that confer a strong selective advantage upon the bacterial clone in maintaining chronic infection. We have previously tested the pathogenic potential of nineteen early and late P. aeruginosa isolates from six CF patients in a murine model of chronic infection. In vivo experiments demonstrated that independent of the genetic background of the clone, adaptation of P. aeruginosa to the CF airways drives towards a pathotype that is attenuated in its acute virulence, but is capable to persist. Pathogenicity-adaptive mutations responsible for favouring P. aeruginosa chronic infection are partially known. Here, we searched for P. aeruginosa pathogenicity-adaptive mutation by exploiting a new positive selection Signature Tagged Mutagenesis (STM) approach. A collection of

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6912 P. aeruginosa PAO1 mutants was screened in the agar beads mouse model which mimics the chronic infection as in patients with CF. Consecutive screenings were carried out which reduced to 16 the P. aeruginosa mutants with a selective advantage. Surviving mutants were recovered and processed for gene identification. We identified insertions in virulence genes implicated in motility and attachment, amino acid biosynthesis and metabolism, secreted factors and transport system and coding for hypothetical or unknown proteins. One insertion was found in an intergenic region. Then we screened our panel of P. aeruginosa STM mutants for phenotypic mutations. Loss of twitching or swimming motility was observed in five strains. Different capacities to produce pyocyanin and form biofilms were observed in two and four strains, respectively. Next, P. aeruginosa STM mutants were tested for invasion in A549 cells. Twelve mutants invaded significantly more the epithelial cells when compared to the reference strain PAO1; while one mutant invaded less. Validation of our panel of genes in terms of genetic diversity in P. aeruginosa clinical strains of different origins is under investigation. These results contribute to establish a new model directed to identify novel bacterial functions involved in P. aeruginosa lung pathogenesis.Supported by Italian CF Research Foundation and Fondazione Cariplo.


TnpR of ISPpu12 regulates transposition of ISL3-like insertion sequences in Pseudomonas stutzeri AN10Rafael Bosch1, Joseph Alexander Christie-Oleza1, Jorge Lalucat1 and Balbina Nogales1

1 Universtitat de les Illes Balears, Palma de Mallorca, Spain

We have recently characterized a novel insertion sequence (IS) named ISPst9 from the naphthalene degrading bacterium Pseudomonas stutzeri AN10 (1, 2). ISPst9 is a 2.5-kb IS of the ISL3-family that is flanked by 24 bp inverted repeats and generates 8-bp AT-rich target duplication upon insertion (1). We demonstrated that ISPst9 transposes by excision from its donor DNA using a conservative (cut-and-paste) mechanism (2). After excision, and prior to insertion in the receptor DNA, ISPst9 is stabilized as a double-stranded DNA circular intermediate (2). Surprisingly, transposition of ISPst9 was induced in all P. stutzeri AN10 cells after conjugative interaction with Escherichia coli, being this a novel and strong IS regulation stimulus never reported before (2).As Southern blot analysis revealed the presence of a second copy of ISPst9 in P. stutzeri AN10 (2), we decided to identify and characterize it in order to evaluate the possibility that both ISPst9 copies constituted a composite transposon. In this report we present a ISPpu12 isoform as the second ISPst9 copy observed in P. stuzeri AN10. ISPpu12 is another ISL3-like insertion sequence that has been extensively characterized (3, 4). Although both IS do not constitute a composite transposon, we have demonstrated that the ISPpu12 of P. stutzeri AN10 transposes using a conservative mechanism like ISPst9. As described for ISPst9, the transposition of the ISPpu12 is also up-regulated in all cells that suffer conjugative interaction. We have focussed our work on trying to understand how the stimulus (conjugative interaction) up-regulated IS transposition. We have demonstrated that transposition activation after stimulus is exclusive of the ISPpu12, although transposase activity of this IS can act in trans on ISPst9, increasing transposition of both ISs. Furthermore, we have shown that transposition of both ISs is not produced when tnpR, the MerR-like transcriptional regulator encoding gene present in the ISPpu12, is deleted. Therefore, we hypothesize that conjugative interaction activates TnpR of the ISPpu12 isoform. TnpR activation would eventually cause a transcriptional increase of the ISPpu12 transposase, resulting in an elevated IS transposition.References.1. Christie-Oleza et al. (2008) Int Microbiol 11: 101-1112. Christie-Oleza et al. (2009) J Bacteriol 191: 1239-12473. Weightman et al. (2002) J Bacteriol 184: 6581-65914. Williams et al. (2002) J Bacteriol 184: 6572-6580


Identification of a putative operon involved in mangotoxin biosynthesisFrancisco M. Cazorla1, Victor J. Carrión1, Jose A. Gutiérrez-Barranquero1, Eva Arrebola1, Jesus Murillo2 and Antonio de Vicente1

1 Grupo de Microbiología y Patología Vegetal. Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071.,2 Laboratorio de Patología Vegetal, ETS de Ingenieros Agrónomos, Universidad Pública de Navarra. 31006-Pamplona.

The Pseudomonas syringae pv. syringae (Pss) UMAF0158 strain is a phytopathogenic bacterium isolated from mango (Mangifera indica), which causes the bacterial apical necrosis of mango (NAM). This Pss strain produces mangotoxin, an antimetabolite toxin which have the enzyme ornithine acetyltransferase as target, thus inhibiting the conversion of N-acetyl ornithine to L-ornithine and interfering with the ornithine/arginine biosynthesis. In order to search for genes involved in the mangotoxin production, a chromosomal region of 11.5 kb from Pss UMAF0158 was obtained and cloned (pCG1-6). This chromosomal region included the gene mgoA, which is a non-ribosomal peptide synthetase involved in the mangotoxin biosynthesis; however, its only presence is not sufficient for mangotoxin production.Simultaneously, a random Tn5 mutant library was constructed, selecting some derivative strains impaired in the mangotoxin production. In this work, we started the study of two of these Tn5 mutants (disrupting homologous genes to a caboxylase and amidino transferase, respectively). Using these disrupted sequences as probes, a new chromosomal region of 14 kb was obtained (cloned in pCG1-5) from a genomic library of Pss UMAF0158, which can revert mangotoxin production in these two defective Tn5 mutants. The pCG1-5 contains a total of twelve ORFs; six ORFs, also present in the sequenced non mangotoxin-producing strain Pss B728a, are flanking a group of six other ORFs, which are not detected in any of the sequenced Pseudomonas. These six ORFs included those disrupted by the two different Tn5 mutants.To elucidate the function of the six ORFs, site-directed mutagenesis using the commercial vector pCR2.1® was carried out. Currently, six mutants in each ORF of this cluster have been constructed, and the alteration in the production of mangotoxin has been checked by means of Escherichia coli growth inhibition assay. Complementation experiments are currently on-going.This work has been supported by grants from Proyecto de Excelencia P07-AGR-02471.

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Towards a definition of the stress protein interactome of Pseudomonas putidaThorben Dammeyer1, Sagrario Arias-Rivas2, Agatha Bielecka1, Tatyana Chernikova1, Taras Nechitaylo1, Peter N. Golyshin1 and Kenneth N. Timmis3

1 Helmholtz Centre for Infection Research, Braunschweig, Germany, 2 Technical University Braunschweig, Germany, 3 Helmholtz Centre for Infection Research and Technical University, Braunschweig, Germany

Proteins are the principal agents mediating most cellular functions. In general, they do so not as individual entities but rather as higher order multi-protein complexes interacting in a variety of ways. Knowledge of these interactions is essential not only for understanding the function of a cellular system, but also for optimization of its performance in biotechnological processes. Genome sequenced Pseudomonas putida KT2440 is one of the best studied strains of the metabolically versatile and ubiquitous genus Pseudomonas and exhibits wide biotechnological potential due to its stress resistance, amenability for genetic manipulation and suitability as a heterologous expression host. Because industrial applications often impose unnatural stresses upon production organisms, and counteracting such stresses may reduce productivity, our goal is to unravel production-relevant stress networks in the context of a systems biology study of P. putida and identify potential points of intervention in the networks to increase productivity. We first identify by proteomics stress proteins induced by hyperproduction of enzymes. These stress proteins are then used as baits to affinity purify interacting proteins. Knowledge of these interactions will help to assign functions to proteins with unknown function, uncover new metabolic interconnections and identify new points of intervention for the optimization of the cell factory.


Accurate taxonomic assignment for ultra-short metagenomic readsColin F Davenport1, Jens Neugebauer1, Oleg Reva2 and Burkhard Tümmler1

1 Hannover Medical School, 2 Bioinformatics and Computational Biology Unit, Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa

In the emerging metagenomic era new sequencing technologies are set to massively increase the availability of sequence information from environmental sources. In place of single strains, partial sequencing of prokaryotic communities is becoming more viable. Short (36bp) DNA reads from environmental samples are difficult to confidently assign to a species with current bioinformatic tools, and reliable phylogenetic markers occur too infrequently to be useful in identifying strains. Next generation sequencers produce the highest volume of reads and lowest cost per base pair, and are thus particularly suitable for sampling diverse microbial communities. We aim to use short reads to identify species within a community via two distinct computational approaches.The first approach uses the overrepresented oligomer words derived from completely sequenced genomes by the program OligoCounter. These words are informative for bacterial genome structure, carry a phylogenetic signal and may be characteristic for individual bacterial genomes. Particularly common and widely distributed yet genome specific oligomers are termed markers. These markers are then used in a fast two stage procedure to assign metagenomic reads to genus or species level taxonomic groups.The second approach uses the power of current short read mapping tools. Short reads, or short tags from longer reads, can be very quickly mapped onto a metagenomic reference. Mapped reads are then counted, taxonomy is assigned and they are graphically presented. This method may even allow strain level identification for some species.These methods show considerable promise, with algorithms correctly identifying the most common species within a 10-species artificial metagenome. The very low number of false positives from the read mapping approach is particularly encouraging. We aim to soon introduce a web service to enable identification of short orphan environmental sequences with appropriate confidence statistics, for use in medical, industrial or academic contexts.


Novel microarray and short read sequencing technology revealing the genetic background of phenotypic variants in P. aeruginosa Andreas Dötsch1, Tanja Becker1, Michael Jarek2, Maren Scharfe2, Robert Geffers1, Helmut Blöcker2 and Susanne Häussler1

1 Cell and Immune Biology, Helmholtz Center for Infection Research, Braunschweig, Germany, 2 Genome Analysis, Helmholtz Center for Infection Research, Braunschweig, Germany

Pseudomonas aeruginosa owes most of its capability to thrive under a vast variety of environmental conditions to its modular genome and phenotypic adaptability. In chronic infections - such as in patients suffering from cystic fibrosis - P. aeruginosa easily adapts to its environment by developing variants that show increased resistance towards the host immune defense and antimicrobial treatment.Clinical and laboratory experiments have already shown that P. aeruginosa can form a genetically diverse community generating an 'insurance effect' by rendering the population more flexible towards different environmental conditions. Since the underlying genetic variations range from insertion or deletion of large parts of the chromosome down to single nucleotide polymorphisms (SNPs), it is both essential and challenging to determine the complete genotype. Just recently, major advances in DNA-microarray and sequencing technologies enabled the analysis of complete genomes with a reasonable effort in time and money.We have isolated three distinct morphological variants occurring reproducibly after a few days of incubation of P. aeruginosa PAO1 on soft agar plates. These variants exhibit large differences in motility and biofilm formation and remain stable under different culture conditions suggesting that this variation might be caused by genetic differences.However, comparative genome hybridization (CGH) of the three variants with a recently described PAO1 whole genome DNA-microarray (PATA1) did not detect any genetic variation. Potential small genetic differences might have been missed, because of the limitations of PATA1 (SNP detection with ~ 50 % sensitivity).Therefore, one of the variants was selected and analyzed with an Illumina Genome Analyzer. Indeed, a single nucleotide exchange could be detected within the type IV fimbrial precursor gene pilA (PA4525). This allele was subsequently confirmed to be present in 13 independent isolates suggesting a strong link between allele and phenotype.We demonstrate the applicability of microarray based comparative genome hybridization and next generation sequencing to unravel the genetic determinants of phenotypic variants.

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Proteome analyis of Pseudomonas putida response to overexpression of a heterologous lipaseHorst Funken1, Karl-Erich Jaeger1, Susanne Wilhelm1 and Frank Rosenau1

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, at Forschungszentrum Juelich, Germany

Overexpression of heterologous proteins often poses cellular stress to the host cell. We have studied the molecular consequences of expression-induced stress in the biotechnological relevant host strain Pseudomonas putida KT2440 by analyzing its cellular proteome while overexpressing the lipase operon lipAH from P. aeruginosa. Specifically, the stress response of P. putida was investigated by determination of growth, lipase protein expression, activity and 2D-gel electrophoretic separation of cellular proteins including analysis with the "delta 2D"-software (DECODON, Germany) and protein identification by MALDI - TOF mass spectrometry. A total number of 1500 P. putida proteins were identified and significant differences in spot intensities were detected upon overexpression of the lipase operon. About 120 proteins were found to be either down- or upregulated in the lipase overexpression strain. Mass spectrometric identification revealed that several outer membrane proteins were produced in significantly increased amounts under lipase overexpression conditions, whereas some transcriptional regulators where found decreased.


Role of the Pseudomonas aeruginosa minor pilins in type IV pilus biogenesis and type II secretion systemsCarmen L. Giltner1, Marc Habash2 and Lori L. Burrows1

1 McMaster University, 2 University of Guelph, Canada

Assembly of type IV pili (T4P) in Pseudomonas aeruginosa requires a number of proteins including the major pilin (PilA) and several structurally related minor pilins encoded by the fimUpilVWXE operon. Disruption in any of the minor pilin genes abolished twitching motility and surface piliation, which was complemented by the cognate gene under an arabinose inducible promoter. Overexpression of any of the minor pilin genes with higher levels of arabinose reduced motility and surface piliation without affecting intracellular PilA levels. To determine if these genes are essential for assembly the PilT retraction ATPase was disabled in each of the minor pilin mutants; in each case surface piliation was restored indicating that the minor pilins are not essential for initiation of pilus assembly.The T4P and type II secretion (T2S) systems share a similar architecture and many homologous proteins, suggesting a common evolutionary ancestor. Pseudomonas aeruginosa uses at least 3 different T2S systems to export enzymes and toxins from the bacterial periplasm to the extracellular milieu. With the exception of the prepilin peptidase PilD, inactivation of genes involved in T4P biogenesis does not affect T2S and vice versa, suggesting that the two types of complexes are distinct entities. Unexpectedly, minor pilin mutants disrupted Xcp-mediated T2S of specific effectors, while overexpression of the minor pilin proteins FimU and PilX increased T2S effector secretion to levels greater than 3 times wildtype. Further examination showed that effectors dependent upon the Sec system for export across the cytoplasmic membrane (i. e. elastase) were not secreted by the minor pilin mutants, but those dependent upon the Tat export system (i. e. lipase) were unaffected. These data suggest that either the T4P system plays a role in Sec-, but not Tat-dependent secretion of T2S effectors, or that disruption of the putative minor pilin complex impairs Sec-dependent transport of other proteins. The latter scenario is plausible as the major pilin, PilA, inserts into the inner membrane in a Sec-dependent manner. Together, these results show that twitching motility and secretion are complex phenotypes that rely on tight control of the levels of components involved in the assembly process in order to operate within normal parameters, and that the systems have more points of intersection than previously thought.


Diversification and evolution of Pseudomonas aeruginosa in the upper and lower airways of intermittently colonized CF childrenSusse Kirkelund Hansen1, Helle Krogh Johansen2, Rasmus Bojsen1, Jakob Frimodt-Møller1, Niels Høiby2 and Søren Molin1

1 Infection Microbiology Group, Technical University of Denmark, DK-2800 Lyngby, Denmark, 2 Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark

Pseudomonas aeruginosa lower airway infection is an important cause of morbidity and mortality in cystic fibrosis (CF). During the cause of infection P. aeruginosa is known to evolve and adapt to the CF lung. Upper airway infection leading to chronic sinusitis is a common complication in CF that is often seen from a very early age. However, the role of the paranasal sinuses as a possible reservoir for recolonization of the lung and as a focus for initial diversification and evolution has received little attention. The objective of this study was to perform a genotypic and phenotypic comparison of P. aeruginosa populations isolated from upper and lower airways in intermittently colonized CF children. Seven children that had been treated with Functional Endoscopic Sinus Surgery (FESS) were selected for this study. Samples from the sinuses were collected during surgery but prior to surgery the infection history of the lower airways had been monitored by collecting successive sputum samples obtained by expectoration or endolaryngeal suction. In each CF child the upper and lower airways were colonized by an identical but unique clonal line of P. aeruginosa as based on the SNP pattern using the AT-Chip (Clondiag). A high degree of morphological diversity (based on colony morphology) was observed for populations from the paranasal sinuses and often the left and right side of the sinuses had evolved morphologically distinct phenotypes. For each patient a subset of identical morphotypes were found in both the upper and lower airway populations. A phenotypic profile was scored for the different colony morphologies. The shared morphotypes had an identical or very similar phenotypic profile suggesting a common origin. Interestingly, the results of the phenotypic profiles showed that several important traits in relation to the chronic lung infection had in fact evolved in the sinuses: reduced motility, mutation in the las quorum-sensing system, reduction in virulence production, changes in biofilm formation in addition to the appearance of mucoid and small colony variants. Our results indicate that the paranasal sinuses are a diversifying, compartmentalized niche from where the traffic of P. aeruginosa is mainly downwards. The upper airways are likely a reservoir of P. aeruginosa for recolonisation of the lower airways and the paranasal sinuses may play a significant role for some patients in the initial evolution of chronic lung infection.

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Colonization of barley roots by Pseudomonas putida is influenced by FisJulia Jakovleva1, Anna Velts1, Annika Teppo1, Maia Kivisaar2 and Riho Teras1

1 Institution of Molecular and Cell Biology, Tartu University and Estonian Biocentre, Riia mnt 23, Tartu 51010, Estonia

Fis (factor for inversion stimulation) is a small homodimeric nucleoid-associated bacterial protein that is well known from studies of Escherichia coli as a trigger of fast growth of bacteria under conditions of abundance of nutrients. Fis regulates transciption of large numbers of genes and thereby can serve as a very important link between signals from the environment and bacterial physiological state. Very little is known about the functions of Fis in Pseudomonas putida, a soil bacterium able to colonize plant roots. The aim of our studies was to investigate whether Fis could influence colonization of barley roots (Hordeum vulgaris) by Pseudomonas putida strain PaW85.We constructed P. putida strains either over-expressing Fis (strain F15) or reducing Fis expression by anti-sense RNA (strain FB) and studied ability of these strains to colonize barley roots in comparison with the wild-type strain. 4 days pre-germinated barley seedlings were inoculated either with pure culture of cells of F15 or FB or in mixture with wild-type cells. We estimated persistence of each strain on roots alone or in competition experiments during 21 days. Cells of F15 and FB were tagged by gentamycin resistance gene and cells of wild-type strain PaW85 by streptomycin resistance gene. We found that cells of P. putida wild-type strain effectively out-competed F15 cells over-expressing Fis but not those FB cells where the expression of Fis was artificially reduced. Taking together, the results obtained in this study indicate that Fis participates in regulatory network influencing the ability of P. putida to colonize plant roots.


Signature-tagged mutagenesis identifies newly introduced genomic prophage islands as critical determinants of in vivo competitiveness in Pseudomonas aeruginosa LESB58.Irena Kukavica-Ibrulj1, Dr Catherine Paradis-Bleau2, Dr François Sanschagrin3 and Dr Roger Levesque1

1 Institut de Biologie Intégrative et des Systèmes (IBIS), Laval University, Québec, Canada, 2 Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, 3 Service d'Anatomo-Pathologie, Hôpital du Saint-Sacrement, Québec, Canada

Pseudomonas aeruginosa isolates have a highly conserved 6.4 Mb core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands (GI). P. aeruginosa Liverpool Epidemic Strain (LES) is an aggressive strain originally identified in the UK but now isolated in Canada from CF patients. To learn about the forces that shaped the development of this important epidemic strain, we performed a signature-tagged mutagenesis (STM) screen using 9,216 STM mutants in a rat model of chronic lung infection. After 7 days post infection, STM screening identified 163 STM mutants as defective for in vivo maintenance. DNA sequencing revealed insertions in most known functional gene classes. These included insertions in genes encoding products or processes implicated in pathogenesis of P. aeruginosa, such as the type III secretion protein PscH, a haem iron uptake receptor PhuR, TolA, the fimbrial usher CupA3, the alginate biosynthesis protein MucD, and transcriptional regulators. Insertions in genes involved in the biosynthesis of type III pyoverdine (pvdE) and pyochelin were identified, emphasizing the importance of both siderophores. To assist in understanding the basis for the successful colonization of the LES in CF patients, the level of attenuation in vivo was determined for 9 STM mutants having insertions in LES prophages 1, 2, 3, 5 and 6 and 3 STM mutant in the unique LESGI (LESGI-4 and 5). Mutants with insertions in three prophages (1, 3 and 5) or in LESGI-5 caused a severe defect in growth and maintenance in vivo, which gave a significant 7- to 1000-fold decrease of CFUs in rat lung tissues with CI values of 0.001 to 0.15. This indicated that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of genomic prophage genes.


Transcriptome response to nitric oxide in Pseudomonas aeruginosaMiho Kuroki1, Hiroyuki Arai1, Masaharu Ishii1 and Yasuo Igarashi1

1 University of Tokyo, Japan

Nitric oxide (NO) is a toxic molecule known to be produced by macrophages in the host immune defense mechanism against pathogenic bacteria. It is also produced as an intermediate of bacterial denitrification. The opportunistic pathogen Pseudomonas aeruginosa has two NO detoxification enzymes flavohemoglobin (Fhp) and NO reductase (NOR). Fhp oxidizes NO to nitrate under aerobic conditions. NOR is one of the denitrification enzymes and reduces NO to nitrous oxide under anaerobic conditions. Two NO-sensing transcriptional regulators, FhpR and DNR, independently regulate the expression of Fhp and NOR, respectively. DNR is a specific regulator for denitrification and its expression is under the control of ANR, which is a global regulator for anaerobic gene expression in P. aeruginosa. Both ANR and DNR are members of the CRP/FNR-family of transcriptional regulators and have overlapping regulons. ANR is a homologue of Escherichia coli FNR, which is basically an oxygen sensing regulator but is known to have an ability to respond to NO because its oxygen-sensing Fe-S cluster is sensitive to NO. Therefore, FhpR, DNR, and ANR are expected to have important roles in the protection of P. aeruginosa against NO. In this study, we investigated the roles played by these regulators in the transcriptome response to NO in P. aeruginosa.Semiaerobic cultures of PAO1 and its isogenic fhpR, dnr, and anr mutant strains and an anr mutant modified to constitutively express dnr were exposed to NO by adding NO-saturated water. RNA samples were extracted from the cells before and after the exposure to NO and subjected to microarray analysis. FhpR was revealed to regulate only the fhp-ppyR-nnrS operon. DNR was revealed to specifically regulate the denitrification-related genes such as nir, nor, and nos genes for nitrite, NO, and nitrous oxide reductases, respectively. Some genes including the heme biosynthesis genes were found to be regulated by both ANR and DNR. The nir genes were mainly upregulated by DNR in response to NO and also weakly activated by ANR in the absence of NO. The other denitrification-related nor and nos genes were not directly regulated by ANR. Most of the other NO-responsive genes, whose regulation was independent from FhpR, DNR, or ANR, are known to be upregulated under iron-limiting conditions and probably in the regulon of ferric uptake regulatory protein Fur, suggesting that Fur is also involved in the NO-sensing regulation in P. aeruginosa.

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Pseudomonas putida KT2440: genome minimization projectAudrey Leprince1, Kenneth Nigel Timmis2, Victor de Lorenzo3 and Vitor A.P. Martins dos Santos1

1 Systems and Synthetic Biology Group, Helmholtz Zentrum für Infektionsforschung (HZI), 2 Environmental Microbiology Group, HZI, 3 Centro Nacional de Biotecnología-CSIC, Madrid, Spain

Synthetic biology can be used as an improvement tool in the process of biotransformation of chloroaromatic compounds. Based on the 6.18 Mb genome of Pseudomonas putida KT2440, and its 5347 genes, our experiments aim at the construction of a functioning, streamlined bacterial cell devoid of different parts of its genome and endowed with a series of highly coordinated, newly assembled genetic circuits. The reduction of the genome carried out under given conditions will allow the identification of the essential gene set that is necessary and sufficient to sustain the functioning of a cell. The second step consists of the creation of a biocatalyst, under the same conditions, capable of degrading compounds such as toluene or benzoate into high value compounds, which is of industrial interest. Reducing the physical size of the genome can be considered as an optimization for a better control of the bacterial strain during biotransformation, due to improved knowledge.The gene deletion process is based on two different approaches. The first approach consists in deleting a single target gene or a group of genes. Fragments of interest meaning DNA fragments non essential for the survival of the bacteria under specific conditions are selected by computational analyses: comparative genomics or analysis of the GC and CAI contents of P. putida. A series of suicide plasmids is now constructed and ready to use for single homologous recombination events. In the second approach fragments have to be randomly removed from the chromosome, which allows a faster reduction of the number of genes within less manipulations. A set of two modified mini-Tn5 transposons is randomly inserted in the chromosome of P. putida and a library of clones is under construction. The Saccharomyces cerevisiae Flp-FRT recombination system is used in both methods as the final step allowing deletions.Fragments of interest will be removed allowing better manipulation of the strain for the insertion of new genetic circuits in the genome. Random deletions will identify new functions for hypothetical and conserved hypothetical protein. Genotypic and phenotypic analyses are part of the process in order to follow precisely the gene deletions and the consequences on the behavior of the bacteria in the described medium.


Characterization of two clonal strains of Pseudomonas aeruginosa in Cystic Fibrosis patients in Ontario, CanadaTracy Lewis1, Karam Ramotar1, Eleanora Altman2, Jianjun Li2, Kathy Vandemheen1, Wendy Ferris3 and Shawn Aaron1

1 Ottawa Health Research Institute, 2 National Research Council - Institute for Biological Sciences, 3 Children's Hospital of Eastern Ontario, Canada

Chronic infection of the airways with Pseudomonas aeruginosa (PA) occurs in up to 60-70% of Canadian adults with Cystic Fibrosis (CF). Differences in clinical outcomes amongst patients may be partly due to differences among infecting strains of PA. We studied PA isolated from sputum from 425 adult CF patients in Ontario, Canada as part of a four-year prospective cohort study of all adult CF patients in the province. We used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and lipopolysaccharide (LPS) analysis of the bacterial cell envelope to genotype and characterize PA isolates.PA was isolated from 309 adult CF patients in Ontario. Isolates from each patient were analyzed by PFGE to determine molecular genotype. Thirty-two of these isolates were selected based on their PFGE type for MLST analysis. MLST was performed by amplification and sequencing of seven housekeeping genes and results were compared to the PA MLST database. Thirty of the 32 isolates were further analyzed by capillary electrophoresis-mass spectrometry to determine LPS profiles. Antimicrobial susceptibility testing was also performed on these 30 isolates.Using PFGE we found 2 clonal strains of PA among the CF population in Ontario, currently named strain A with 22% prevalence and strain B with 11% prevalence. Using MLST we were able to differentiate between these two strains and other unique PA strains. Strain A was compared to the PA MLST database and was a match to the Liverpool strain (ST-146) in the United Kingdom. Strain B (ST-439) has not been previously described as a clonal strain. LPS analysis also differentiated between the two clonal strains and the unique strains. Strain A was identified as having a smooth-type LPS phenotype containing O-chain polysaccharide and strain B a rough LPS phenotype. Susceptibility testing indicated strains A and B were less susceptible to many antibiotics compared to the unique strains.In conclusion, we have found two clonal strains of PA infecting adult CF patients in Ontario; one strain appears to be the Liverpool strain and the other has not been previously described. These strains can be differentiated from each other, and from unique strains of PA, using PFGE, MLST and mass spectrometry techniques.


Genetic and structural characterization of a new family of secondary siderophores and a phylogenetic study of its widespread distribution across Pseudomonas spp.Sandra Matthijs1, Vittoria Catara2, Georges Laus3, Herbert Budzikiewicz4 and Pierre Cornelis5

1 Free University of Brussels, 2 Dipartimento di Scienze e Tecnologie Fitosanitarie, Universita di Catania, via S. Sofia 100, 95123 Catania, 3 Laboratory of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, 4 Institut für Organische Chemie der Universität zu Köln, Greinstr. 4, D-50939 Köln, Germany, 5 Laboratory of Microbial Interactions, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium

To cope with iron deficiency Pseudomonas corrugata produces the lipopeptide siderophore corrugatin (Risse et al., 1998). Recently it was found that several Pseudomonas species produce siderophores structurally related to corrugatin, namely ornicorrugatin (Matthijs et al., 2008) and histicorrugatin, demonstrating the existence of a family of corrugatin-like siderophores. Since the biosynthetic, transport and uptake genes of corrugatin and related siderophores have not been described yet a Tn5 mutagenesis was done on the ornicorrugatin-producing strain P. fluorescens SBW25 and several ornicorrugatin-negative mutants were obtained. Through in silico analysis and the construction of several deletion mutants 3 putative operons comprising 11 ORFs have been identified

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in SBW25. Deletion of putative biosynthetic genes (nrps genes) and the putative receptor led to a complete loss or reduction of siderophore production, respectively.To study the distribution of Pseudomonas strains producing corrugatin-like siderophores a large strain collection of 104 Pseudomonas type strains and 400 strains from different geographical and biological origin was screened for the production of these siderophores by PCR with a specific primer set. The production of the siderophore by PCR-positive candidates was subsequently confirmed by purification and identification of the siderophore. Phylogenetic analysis shows that these siderophores are only produced by members of the fluorescens cluster. Corrugatin production is strain specific, it is only produced by P. corrugata (19 strains analysed) whereas orni- and histicorrugatin producing strains are more distributed across the fluorescens cluster. In addition, it was found that many strains belonging to the fluorescens cluster and 2 species of the putida cluster, which do not produce corrugatin or related siderophores, do posses the receptor enabling them to utilize corrugatin and/or related siderophores. Preliminary phylogenetic analysis indicates that corrugatin and histicorrugatin evolved from ornicorrugatin.Risse D., Beiderbeck H., Taraz K., Budzikiewicz H. and gustine D., 1998. Corrugatin, a lipopeptide siderophore from Pseudomonas corrugata. Z Naturforsch 53c, 295-304.Matthijs S., Budzikiewicz H., Schäfer M., Wathelet B. and Cornelis P., 2008. Ornicorrugatin, a new siderophore from Pseudomonas fluorescens AF76. Z Naturforsch 63c, 8-12.


Interference with partitioning system of IncP-7 plasmids by ParA-like protein from Pseudomonas putida KT2440 genomic islandMasatoshi Miyakoshi1, Yoshiyuki Ohtsubo1, Yuji Nagata1 and Masataka Tsuda1

1 Graduate School of Life Sciences, Tohoku University, Japan

At least 14 incompatibility groups of plasmids can be maintained in Pseudomonads. Many of them are transferred by conjugation between various bacteria, but mechanistic barriers in the recipient cells can limit their host ranges. pWW53 and pDK1 are well-characterized toluene-catabolic plasmids from Pseudomonas putida strains, and belong to the narrow-host-range IncP-7 group. We found that the two plasmids can be stably maintained in several Pseudomonas strains but not in P. putida KT2440, in which the plasmids were immediately lost under non-selective conditions. Since the partitioning ATPase, ParA, of IncP-7 plasmids upregulates the expression of a heterologous parA-like gene (parI) of KT2440 (Miyakoshi et al., 2007. J Bacteriol 189: 6849-6860), we hypothesized that the parI product affects the maintenance of IncP-7 plasmids. Comparative genomic analysis of KT2440 with other Pseudomonas strains revealed that parI is located on a KT2440-specific 60-kb genomic island. Removal of parI from KT2440 restored the stable maintenance of IncP-7 mini-replicons, and expression of ParI in P. fluorescens Pf-5 led to a dose-dependent loss of the mini-replicons. These results indicate that ParI is the primary factor that destabilizes the IncP-7 plasmids, probably by inhibiting the plasmid partitioning system. Although the backbones of pWW53 and pDK1 are highly conserved, the mini-pDK1 containing two parS sites was more stable than the mini-pWW53 containing four parS sites in the ParI-expressing derivative of Pf-5 strain, and further deletion to single parS site in the mini-pDK1 increased the stability. These results demonstrate that the number of parS sites determines the loss rate of IncP-7 mini-replicon by ParI. Given that binding of ParB to parS sites forms a pair of plasmid molecules, ParI might interfere with the dissociation of plasmid pairs after replication, which has been assumed to be critical for the active plasmid partitioning. Mutations in putative ATP-binding motifs of ParI almost abolished the inhibitory effect on the plasmid stability, suggesting that the ATPase activity is necessary. Further biochemical and cytological analyses of ParI will provide new insight into the active partitioning mechanism of bacterial replicons. We propose a theoretical extension of 'partition-mediated incompatibility' to phylogenetically distinct mobile genetic elements.


Genetic determinants of Pseudomonas aeruginosa biofilm developmentMathias Müsken1, Stefano Di Fiore2, Andreas Dötsch1 and Susanne Häussler1

1 Cell and Immune Biology, Helmholtz-Center for Infection Research, Braunschweig, Germany, 2 Institute for Molecular Biotechnology, RWTH Aachen University, c/o Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany

The establishment of bacterial biofilms on surfaces requires a complex developmental process that is largely influenced by environmental as well as bacterial signals. Here we report the screen of the comprehensive Harvard Pseudomonas aeruginosa PA14 mutant library for mutants exhibiting an altered biofilm phenotype. We analysed the capability of all mutants to form mature biofilms at the bottom of a 96-well plate by the use of an automated confocal laser scanning microscope and uncovered 177 and 109 genetic determinants of reduced and enhanced biofilm production, respectively. Overall, more than 65 % of the identified mutants were affected within genes encoding for hypothetical proteins, presuming that novel developmental pathways are likely to be involved in biofilm formation. Nevertheless, a common theme that emerged from the analysis of the genes with a predicted function, is that the establishment of a biofilm requires regulatory components that are involved in survival under microaerophilic growth conditions as well as the maintenance of arginine metabolism and pH homeostasis.


Role of RND efflux pumps in biofilms of Pseudomonas aeruginosaSanaya Z Patell1, Nick Bond2, Kathryn Lilley2 and Martin Welch3

1 University of Cambridge, 2 Cambridge Centre for Proteomics Cambridge System Biology Centre University of Cambridge Department of Biochemistry Tennis Court Road Cambridge CB2 1QR, 3 Department of Biochemistry, University of Cambridge - CB2-1QW, UK

Pseudomonas aeruginosa is an opportunistic human pathogen. Infections caused by P. aeruginosa are often exceptionally difficult to treat due to the multi-drug resistant nature of the organism. The origin of this exceptional multi-drug resistance became clear when the P. aeruginosa genome was sequenced; the bacterium encodes no less than 12 RND-type efflux pumps. Using global transcriptomic approaches, previous workers have found that certain RND efflux pumps appear to be differentially-expressed in P. aeruginosa biofilms (compared with their planktonic counterparts), raising the possibility that RND pump expression may account for the exceptional antibiotic resistance associated with these sessile structures. However, there appears to be no common thread linking the results of these earlier studies. Therefore, the aim of the current study is to systematically investigate the possible contribution of RND pump expression towards the increased antibiotic resistance of P. aeruginosa biofilms.

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Using a state-of-the-art targeted proteomic approach called QconCAT/MRM-MS we are examining how the RND efflux pump expression profile is affected by growth of P. aeruginosa biofilms and planktonic cells on different nutrient sources, oxygenation, by exposure to antibacterial agents etc. By monitoring the amount of specific RND pump proteins our approach enables quantitation of the physiologically-relevant molecular species in the cell – the pumps themselves - and therefore represents a significant advance on earlier approaches that relied upon quantitation of the corresponding transcripts. Moreover, our approach allows absolute quantitation (on a molecules per cell basis) of the RND proteins, enabling the facile comparison of datasets across time and between different labs. Proteins from planktonic or biofilm cultures of PAO1, along with a selection of multi-drug-resistant clinical isolates from the lungs of cystic fibrosis patients are being analyzed using QconCAT/MRM-MS. For example, our data show pronounced up-regulation of the MexGHI-OpmD pump in wild-type biofilms. In addition, we are also investigating how the global RND pump profile is affected by exposure to a range of clinically-relevant antibiotics and non-antibiotic “inducers” of RND pumps expression, and these results will be presented.


Subinhibitory concentrations of colistin induce the Pseudomonas Quinolone Signal in Pseudomonas aeruginosaJerry Reen1, Joanne Cummins1, Christine Baysse2, Marlies Mooij1 and Fergal O'Gara1

1 BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland, 2 Equipe DUALS UMR6026, Université de Rennes, Campus de Bealieu, F-35043, Rennes, France

Infections with Pseudomonas aeruginosa are particularly difficult to cure through antimicrobial therapy because of the bacterium’s intrinsically impermeable outer membrane and active efflux of toxic agents from the cytoplasm. This has led to renewed interest in an old class of antibiotics known as the polymyxins, first discovered in the 1950’s in the soil bacterium Bacillus polymyxa. Increasingly being used as a salvage therapy, the emergence of resistance to this class of antimicrobial compound would have serious implications for the development of future therapeutic strategies. Indeed, concerns have already been raised about colistin monotherapy, not least with the emergence of heteroresistance among Gram negative pathogens. The effects of subinhibitory concentrations of colistin on gene expression in P. aeruginosa were investigated by transcriptome and functional genomic approaches. Analysis revealed altered expression of 60 genes representing a variety of pathways associated with virulence and bacterial colonisation in chronic infection. These include response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing. Most striking is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Induction of this central component of the QS-network following exposure to subinhibitory concentrations of colistin, may represent a switch to a more robust population, with implications for the successful development and application of colistin therapies in the future.


Allostery in effector secretion control by the P. aeruginosa type III secretion systemArne Rietsch1, Pei-Chung Lee2, Charles Stopford2 and Amanda Svenson2

1 Case Western Reserve University, 2 Dept. of Molecular Biology and Microbiology, 10900 Euclid Ave., Cleveland, OH 44106-4960, USA

Type III secretion systems export effector proteins in a cell-contact dependent manner. Their export prior to cell-contact is controlled by two (in some species three) sets of proteins: the needle-tip protein and an associated regulator (PcrV and PcrG, in the case of P. aeruginosa) and an InvE/YopN-type homolog (PopN). How these proteins control effector secretion is generally unclear.We have analyzed the role of PcrV in controlling effector secretion in P. aeruginosa. A non-secreted version of PcrV lacking its export signal fails to control effector secretion. Replacing the secretion signal with that of ExoS restored effector secretion control and cytotoxicity, indicating that the fusion protein can assemble into a functional needle-tip complex. In the related LcrG-LcrV system of Yersinia sp., the interaction between LcrG and LcrV is required for effector secretion control. Here we show that mutants of PcrV or PcrG that abolish the PcrV-PcrG interaction do not interfere with effector secretion control. Rather, a mutant PcrV that was still able to bind to PcrG, be secreted, but failed to assemble into a functional needle-tip complex lost the ability to control effector secretion.Taken together, our data suggests that PcrV prevents effector secretion prior to cell contact by assembling at the needle-tip, thereby shifting the conformation of the secretion apparatus into the effector secretion "off" state.


Translational stress-induced mutagenesis in Pseudomonas aeruginosaDinesh Diraviam Sriramulu1 and M. Zafri Humayun2

1 CIBIO, University of Trento, Via delle Regole 101, Mattarello (TN) 38060, Italy, 2 Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey – New Jersey Medical School, 225 Warren Street, Newark, NJ, USA

Aminoglycosides are among the most effective antibiotics used worldwide for treating gram-negative bacterial infections, and are also effective against certain gram-positive bacteria. Emergence of aminoglycoside-resistant mutants has become a common phenomenon when pathogenic bacteria are exposed to these antibiotics. The possibility that such exposure can accelerate the emergence of resistant mutants has been underexplored. Genetic defects in bacteria that promote mistranslation induce a mutator phenotype, termed translational stress-induced mutagenesis (TSM), that has been well-studied in Escherichia coli. The TSM process raises the possibility that aminoglycosides, which are known to induce mistranslation, may also induce a mutator state that accelerates the emergence of resistant mutants. Here we show that exposure of P. aeruginosa cells to sublethal concentrations of tobramycin and amikacin, two clinically relevant aminoglycosides, elevates spontaneous mutagenesis, as does the expression of a missense suppressor tRNA encoded by the mutA gene. Exposure to aminoglycosides concomitantly increased mistranslation, suggesting that the mutator phenotype is mediated by the TSM process. The understanding of TSM process will ameliorate the existing knowledge on the emergence and evolution of antibiotic resistance in bacteria. On the other hand, new concerns might arise regarding novel therapies based on mistranslational suppression of genetic defects by aminoglycosides.

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Denitration of 2,4,6-Trinitrotoluene in Aqueous Solutions Using Small-Molecular-Weight Catalyst(s) Secreted by Pseudomonas aeruginosa ESA-5Benoit Stenuit1 and Spiros N. Agathos1

1 Catholic University of Louvain, Unit of Bioengineering, Place Croix du Sud, 2/19 1348 LOUVAIN-LA-NEUVE BELGIQUE

The denitration of 2,4,6-trinitrotoluene (TNT) can produce mono- or dinitro aromatic compounds susceptible to microbial mineralization. In the present study, denitration of TNT and other nitro aromatic compounds was investigated with a solid-phase extract obtained from the culture supernatant of Pseudomonas aeruginosa ESA-5 grown on a chemically defined aerobic medium. When the C18 solid-phase extract containing extracellular catalysts (EC) was incubated with TNT and NAD(P)H, we observed a significant release of nitrite. The concentration of nitrite released in the reaction medium was strongly dependent on the concentration of NAD(P)H and EC. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde, and 2,4,6-trinitrobenzyl alcohol. During this process, the production of toxic reduced TNT metabolites (e.g., aminodinitrotoluene isomers) was minimal. The incubation of EC with TNT, NAD(P)H, and specific scavengers of reactive oxygen species suggested the involvement of superoxide radicals (O2-) and hydrogen peroxide in the denitration process. Results obtained in this study reveal for the first time that extracellular small-molecular-weight substance(s) of bacterial origin can serve as green catalyst(s) to initiate TNT denitration. The protein-free small MW catalyst(s) are released by the environmental isolate P. aeruginosa ESA-5 during stationary phase, i.e., when they face starvation conditions as in environmental habitats. The identification of the catalyst(s) responsible for the denitration of TNT in the presence of NAD(P)H, the determination of their physiological role(s), and the characterization of the underlying reaction mechanism of denitration are currently under way.


Fis negatively regulates binding of Tn4652 transposase to the left end of Tn4652Riho Teras1, Julia Jakovlev12 and Maia Kivisaar1

1 Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, Riia mnt 23, Tartu 51010, Estonia

Transposition of mobile DNA elements is generally random and its activity is maintained at a low level in bacteria. The activity of transposable elements can be controlled by bacterially encoded global regulators. One such example is the regulation of transposition of Tn4652 in Pseudomonas putida. Activation of transposition of Tn4652 in starving bacteria requires the stationary phase sigma factor RpoS (Ilves et al., 2001) and integration host factor (IHF; Ilves et al., 2004). IHF is involved in two processes of regulation: in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652 thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF. Ilves, H., Hõrak, R. & Kivisaar, M. (2001). J Bacteriol 183, 5445-5448.Ilves, H., Hõrak, R., Teras, R. & Kivisaar, M. (2004). Mol Microbiology 51, 1773-1785.


PA4203, a LysR-type transcriptional regulator (LTTR) acts as an autorepressor and a novel modulator of pyocyanin production in Pseudomonas aeruginosaQing Wei1, Saeed Tarighi1, Miguel Cámara2, Paul Williams2, Daniel Charlier3 and Pierre Cornelis1

1 Microbial Interactions, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, 2 Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK, 3 Genetics and Microbiology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

In Pseudomonas aeruginosa, quorum sensing regulates myriads of phenotypic traits associated with virulence via the production of N-acyl homoserine lactones as well as Pseudomonas quinolone signal (PQS). In our previous work, we showed that PA4204 gene in Pseudomonas aeruginosa strain PAO1, encoding a periplasmic gluconolactonase (PpgL), could influence the production of quorum sensing signal molecules. Upstream of ppgL, PA4203, a LysR-type transcriptional regulator (LTTR), was proved to repress expression of its own gene in a luxCDABE-reporter assay. Mutation of PA4203 resulted in serious cell autolysis, confirmed by formation of iridescent sheen and plaque-like clearing on casamino acid media (CAA), and the release of genomic DNA into liquid cultures. Swarming and swimming motilities were also increased in that mutant compared to the wild type. Interestingly, overexpression of PA4203 or complementation of PA4203 into mutant led to overproduction of blue-green pigment pyocyanin on Pseudomonas P-agar, showing PA4203 probably acts as a novel modulator of pyocyanin production by interacting with quorum sensing system(s). Transcriptional analysis of pyocyanin synthesis genes are ongoing and further biochemical analysis by electrophoretic mobility shift assay (EMSA) will be undertaken to identify essential regions responsible for autoregulation and modulation of pyocyanin production. Keywords: LysR-type regulator, regulation, pyocyanin, EMSA


Pseudomonas Genome Database: Facilitating User-friendly, Comprehensive Comparisons of Updated Genome AnnotationsGeoffrey L. Winsor1, Thea Van Rossum1, Leanne Fleming1, Raymond Lo1, Bhavjinder Khaira1, Matthew Whiteside1, Robert Hancock2

and Fiona Brinkman1

1 Simon Fraser University, 2 University of British Columbia, Canada

The Pseudomonas Genome Database (http://www.Pseudomonas.com) was originally developed as a resource for peer-reviewed, continually updated annotation for the Pseudomonas aeruginosa PAO1 reference strain genome. In order to facilitate cross-strain and

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cross-species genome comparisons with other Pseudomonas species of importance, it was expanded to include all completely sequenced Pseudomonas species and has incorporated methods to facilitate high quality comparative genomics. Our advanced search tool provides a flexible, yet user-friendly interface for researchers to retrieve and compare annotations or sequences within or between genomes. To help bridge the gap between applied and basic research, a query for genes that are drug targets will return a list of genes containing relevant drug information and links. Multiple views of the data at sequence and annotation levels are available to facilitate genome comparisons. In addition to new analyses being added to the GBrowse view of genome annotations, a stacked view of orthologs displayed in genomic context with details of how they were assessed is available. To identify large-scale evolutionary events, tools such as Mauve for visualizing whole-genome alignments have been incorporated. Our high-precision method for evaluating previously-predicted orthologs has been complemented with a novel clustering method providing a more inclusive set of genes mapped to their putative orthologs. These Pseudomonas Orthologous Groups correspond better than COGs to what researchers have defined as having a similar gene name. In addition, a new comparative search feature allows ortholog data to be interrogated to retrieve lists of orthologs found (or not found) in two or more organisms. This database aims to continue to provide a high quality, annotated genome resource for the research community by incorporating more than 1800 updates provided by Pseudomonas researchers and in-house curation efforts. Funding provided by Cystic Fibrosis Foundation, Therapeutics, Inc.


Identification of genes involved in swarming motility using a comprehensive Pseudomonas aeruginosa strain PA14 transposon insertion mutant libraryAmy Tsz Yan Yeung1, Ellen Torfs2, Joerg Overhage2, Farzad Jamshidi2, Manjeet Bains2, Irith Wiegand2 and Robert Hancock2

1 University of British Columbia, 2 UBC/232-2259 Lower Mall, Vancouver, BC, V6T 1Z4 , Canada

Besides swimming and twitching, Pseudomonas aeruginosa is capable of swarming on semi-solid (viscous) surfaces. Recent studies have indicated that swarming is a more complex type of motility where swarmer cells exhibit elevated virulence gene expression and antibiotic resistance. During a screening of 5600 mutants from a comprehensive P. aeruginosa PA14 transposon insertion mutant library, 226 mutants were identified with alterations in swarming motility. The identified swarming-associated genes function in diverse processes, including synthesis or function of flagella and type IV pili, antibiotic resistance, regulation, metabolism, chemotaxis, and secretion of virulence factors. Thirty-three swarming deficient and 2 hyper-swarming mutants had transposon insertions in transcriptional regulator genes including those encoding two-component sensors and response regulators; 27 of these were newly identified. Of the 33 regulatory mutants with significant impairments in swarming motility, most had no additional motility defects with 10 mutants showing mild defects (40-50%) and one (PA1458) mutant exhibiting a major swimming defect (91%). Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants, pilH and PA2571. Therefore at least 22 regulatory mutants were identified that were influenced only in swarming motility but not swimming or twitching. Furthermore, some of these regulators are known to regulate critical processes such as carbon and nitrogen metabolism (cbrA, ntrBC) and stress response (cspD). We provide evidence that swarming is controlled by a complex regulatory network and may be part of an alternative growth state for P. aeruginosa. In addition, some mutants that displayed severe swarming defects showed biofilm overproduction while mutants with more moderate deficiencies in swarming showed normal or decreased biofilm formation, indicating an inverse relationship between regulation of these types of adaptations.

Metabolism and Physiology

Oral presentations

O-04 Topic: Metabolism and Physiology

Phenazines control gene expression and community behavior in Pseudomonas aeruginosa PA14Lars EP Dietrich1, Alexa Price-Whelan2 and Dianne Newman2

1 Massachusetts Institute of Technology, 2 M.I.T., 31 Ames Street, Cambridge, MA 02139, U.S.A.

A variety of bacteria are known to release colorful, redox-active pigments. Generally these have been regarded as antibiotics that serve the sole purpose of inhibiting competing organisms. Using RNA arrays we found that one such compound, the endogenous antibiotic pyocyanin (5-methyl-1-hydroxyphenazine) from Pseudomonas aeruginosa PA14, acts as a signaling molecule that activates the transcription factor SoxR. In Escherichia coli, SoxR regulates the superoxide stress response. Surprisingly, bioinformatic analysis coupled with gene expression studies in P. aeruginosa PA14 and Streptomyces coelicolor A3(2) revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses despite the fact that many of these organisms still produce redox-active small molecules. This finding indicated that redox-active pigments play a role independent of oxidative stress. Using P. aeruginosa and S. coelicolor mutants that are defective in biosynthesis of these pigments, we found that they have profound effects on the structural organization of colony biofilms: while wild-type cells form thick and smooth colonies when spotted onto agar plates, the absence of pigments causes the cells to aggregate into visually striking, wrinkled (rugose) morphologies. We are currently testing the idea that rugosity, which results in an increased surface to volume ratio, is an adaptation to electron acceptor limitation. Our results show that redox-active pigments are not merely virulence factors or antibiotics, but also play important, conserved roles in the developmental process of bacterial communities.

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PO-30 Topic: Metabolism and Physiology

Kissing Butterflies and French Fries: The Biosynthesis of Pyocyanin in Pseudomonas aeruginosaWulf Blankenfeldt1, Ekta Ahuja1, Neelakshi Gohain1, Qiang Li1, Petra Janning1, Matthias Mentel2, Almut Graebsch3, Rolf Breinbauer4, Ayten Kalf3, Heide Peters3, Wolf Hiller3, Burkhard Costisella3, Linda Thomashow5 and Dmitri Mavrodi5

1 Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany, 2 Institut für Organische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany, 3 Fakultät Chemie, Technische Universität Dortmund, 44221 Dortmund, Germany, 4 Institut für Organische Chemie, Technische Universität Graz, 8010 Graz, Austria, 5Washington State University, Pullman, Washington 99164-6430, U.S.A.

One of the most conspicuous properties of the human opportunistic pathogen Pseudomonas aeruginosa is the blue-green coloration of late exponential cultures. This effect is due to the excretion of copious amounts of pyocyanin (PYO), a blue phenazine pigment that P. aeruginosa produces under quorum sensing control. The capacity to generate PYO is still used for the identification of P. aeruginosa in the clinic today. It also led to coining the first name of this strain as "Bacillus pyocyaneus" by Gessard in 1882, and is still reflected in the present term: "aerugo" is the Latin word for the blue-green rust of copper. PYO was long believed to act solely as a virulence factor since PYO, like other phenazine derivatives, is redox-active and can shuttle electrons to molecular oxygen, thereby generating toxic reactive oxygen species that contribute to the overall virulence of P. aeruginosa. New data, however, suggest that PYO's importance extends well beyond its toxicity: it may contribute to the acquisition of iron from the environment, could act as an important electron acceptor to reoxidize NADH in the anaerobic layers of biofilm and even has hormone function due to its ability to activate the iron-containing transcription factor SoxR directly. It seems therefore not surprising that defects in PYO production lead to less viable and less virulent strains. Because of its importance, we have investigated the molecular details of PYO biosynthesis by employing structural biology, biochemical analysis and synthetic chemistry. PYO is generated from phenazine-1-carboxylic acid (PCA), which itself is produced from two molecules of chorismic acid in a pathway involving enzymes encoded in the conserved phz-operon. We will show crystal structures of all of these enzymes and give an insight into their mode of action. We will also provide evidence that phenazine biosynthesis, other than implied in the literature, leads to phenazines in their reduced and not in their fully aromatized state. Only in this form can PCA further be converted to PYO, which requires two steps involving the SAM-dependent methyl transferase PhzM and the FAD-dependent monoxygenase PhzS.Our data also show that PYO biosynthesis is accompanied by the generation of considerable amounts of hydrogen peroxide, which seems to require adequate detoxification mechanisms given the high amounts of PYO that P. aeruginosa can produce.


Fine-tuned regulation of rsmY via the HptB phosphorelay pathway in Pseudomonas aeruginosaChristophe Bordi1, Isabelle Ventre1, Marie-Cécile Lamy1, Termine Elis1, Abderrahman Hachani2 and Alain Filloux2

1 CNRS-LISM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France, 2 Imperial College London, Division of Cell and Molecular Biology South Kensington Campus, Flowers Building, SW7 2AZ London, United Kingdom.

Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing either acute or chronic infection in a variety of human infections.The ability of P. aeruginosa to evade the host immune system and to activate virulence mechanisms and therefore to successfully establish infection adequate with the pathogenesis context is strongly dependent on its ability to sense and respond to environmental conditions. Recent studies indicate that signal transduction pathways involving two components systems (TCS) play a key role in the control of P. aeruginosa virulence. It has been shown in particular that the hybrid sensor RetS could belong to a molecular switch promoting expression of the type III secretion machinery (T3SS) often associated to the acute infection and repressing biofilm formation often associated to the chronic infection, whereas an other sensor hybrid sensor LadS acts in an exact opposite way onto these two phenotypes.In this study, we showed that the HptB phosphorelay acts similarly as RetS on biofilm and T3SS. However, despite this similarity, we showed that RetS and HptB do not belong to the same signaling pathway, even though they both intersect with the GacS/GacA pathway. We confirmed that HptB interacts with another response regulator, PA3346, which we named HbsR (HptB-dependent biofilm and secretion regulator). Surpisingly, HptB antagonizes the HbsR activity, since hptB and hsbR mutants have opposite phenotypes. More strikingly, whereas the RetS pathway results in activation of gene expression of both small RNAs rsmY and rsmZ, the HptB pathway only impacts rsmY expression. This crucial difference is adding complexity to the mechanism of regulation through the GacA response regulator, which has thus a different impact on gene regulation depending on the pathway via which it is activated. Furthermore, it shows that the two P. aeruginosa small RNAs rsmY and rsmZ act, by a still unknown process, on common but also on specific targets. Genes encoding the recently discovered Type VI Secretion System might be such specific targets since they are only controlled by RetS and not by the HptB pathway. Further molecular dissection of these complex regulatory events will reveal the powerful fine-tuning possibilities that bacteria developed to respond to tiny environmental changes in a timely and accurate manner.


Regulation of aerobic respiration in Pseudomonas aeruginosa PAO1, evidence for a novel regulatory proteinEmanuela Frangipani1 and Dieter Haas1

1 Department of Fundamental Microbiology, University of Lausanne, Switzerland

P. aeruginosa is a facultative anaerobe with a branched aerobic electron transport chain terminating with five different terminal oxidases. Four of these belong to the haem-copper superfamily and one is the well characterized cyanide insensitive oxidase (CIO), a cytochrome bd quinol oxidase. By controlling the expression of its cytochrome oxidases, P. aeruginosa is able to exploit the most appropriate respiratory pathway according to the environmental conditions. We have recently shown that restricted bioavailability of copper interferes with cellular respiration, being copper an essential cofactor of most terminal oxidases. P. aeruginosa relies on CIO to cope with severe copper deprivation; CIO expression is up-regulated when copper is poorly bioavailable and down-regulated in a copper-replete environment.

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CIO is positively regulated by a two-component system, RoxSR, which is an analogue of the PrrAB system involved in energy conservation in Rhodobacteria. Interestingly, in the prrAB locus there is a third component, named prrC or senC whose product is involved in signal transduction and in cytochrome c assembly. PrrC/SenC might function as a copper chaperone.In P. aeruginosa PAO1, the homologue of prrC/senC is PA0114, which is located apart from roxSR. To investigate the role of PA0114 in the regulation of aerobic cellular respiration, we found that a PA0114 mutant grew slowly during copper shortage, at a rate similar to that of the quadruple cyo cco1 cco2 cox mutant, which is deleted for all known haem-copper oxidases. Moreover, cioAB/PA0114 and roxSR/PA0114 double mutants were severely handicapped in their growth in copper-limiting condition, presumably as a consequence of malfunctioning of the haem-copper terminal oxidases. A cioA'-'lacZ fusion was upregulated in the PA0114 mutant in the absence of copper, compared to the wild-type. These results, together, strongly suggest a role for PA0114 in copper acquisition and in the regulation of aerobic respiration in P. aeruginosa.


Pyruvate dehydrogenase is the metabolic target of the EIIANtr component of the N-related phosphotransferase system (PTSNtr) of Pseudomonas putidaKatharina Pflüger-Grau1 and Víctor de Lorenzo1

1 Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain

Similarly to many other bacteria, Pseudomonas putida KT2440 is endowed with a branch of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTSNtr), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. Dielectric breakdown of P. putida cells grown in complete LB medium revealed the presence of forms of the EIIANtr (PtsN) component of PTSNtr, which were strongly associated to other cytoplasmic proteins. To investigate such intracellular targets of EIIANtr, a soluble protein extract of bacteria bearing an E-epitope tagged version of PtsN was immuno-precipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry. By this approach we could unequivocally identify the E1 subunit of the pyruvate dehydrogenase (PDH) complex (the product of the aceE gene) as a major interaction partner of PtsN (EIIANtr). This enzyme checks inter alia the flux of carbon from catabolism of carbohydrates into the Krebs cycle and thus operates as a divider of metabolic resources into distinct physiological fates. To examine the effect of PtsN (EIIANtr) on PDH, the enzyme activity was measured in extracts of isogenic ptsN+/ptsN- P. putida strains. The results clearly indicated that PtsN (EIIANtr) downregulates PDH activity. Since expression of aceE is not affected by ptsN, our data suggest that PtsN (EIIANtr) controls a major node of C metabolism through direct protein-protein interactions with AceE. These results, together with the observation that the PTSNtr influences expression of the TOL biodegradation pathway, multi-cellular behaviour, morphology, and the intracellular accumulation of polyhydroxyalkanoates, provide evidence for the multiple regulatory functions for the PtsN (EIIANtr) protein of P. putida.


A small RNA of Pseudomonas aeruginosa stimulates the expression of the pyochelin regulatory gene pchRCornelia Reimmann1 and Naomi Borel1

1 University of Lausanne, Switzerland

The siderophore pyochelin is made under iron-limiting growth conditions by Pseudomonas aeruginosa and some strains of the Burkholderia cepacia complex. In P. aeruginosa, the expression of pyochelin biosynthesis and uptake genes strictly depends on the AraC-type protein PchR and pyochelin itself. Upon interaction with the siderophore, PchR binds to conserved sequence elements of target genes and regulates their transcription. For maximal siderophore responsiveness PchR levels need to be low. Under iron-replete conditions this is achieved by Fur-mediated transcriptional repression of pchR, while under iron-limiting conditions pchR is autorepressed by PchR together with pyochelin. Using a genetic screen, we recently gained evidence for a positive control of pchR expression. While a translational pchR-lacZ fusion was expressed under iron-limiting conditions in P. aeruginosa, no expression occurred in Escherichia coli. To identify P. aeruginosa genes that are needed for pchR expression in the heterologous host, a genomic library of P. aeruginosa was transferred to E. coli. Four different clones were identified which activated pchR-lacZ. In all 4 clones the activity was mapped to an intergenic region. According to our bioinformatics analysis, one clone could specify a small regulatory RNA capable of base-pairing with the pchR leader. Small amounts of this RNA, which we have named Rpr16, could be visualized in stationary phase cultures of P. aeruginosa grown under iron limitation. To evaluate the role of Rpr16 in pyochelin biosynthesis and uptake, we are currently investigating the physiological conditions under which Rpr16 is expressed.

P-35 Topic: Metabolism and Physiology

Positive regulation of lipA expression by Hfq in Pseudomonas aeruginosa PAO1Laetitia Abdou1, Elisabeth Sonnleitner1, Sorana Crivii1 and Dieter Haas1

1 University of Lausanne, Department of Fundamental Microbiology, Building Biophore, 1015 Lausanne - Switzerland

Hfq is an RNA-binding protein belonging to the class of Sm-like proteins which acts as a global regulator in numerous Gram-negative bacteria. It has been shown that Hfq contributes to the virulence of Pseudomonas aeruginosa. This bacterium secretes different virulence factors depending on environmental and quorum sensing signals. Among exoenzymes, lipase (LipA) is involved in lung destruction during colonization of the respiratory tract in cystic fibrosis patients. Until now, LipA has mainly been studied for its biotechnological applications but its expression is still not completely understood. LipA is encoded by an operon of two genes. The first gene codes for the structural gene lipA and the second gene lipH codes for the specific foldase of LipA.In Pseudomonas alcaligenes, lipase expression is under the control of the two-component regulatory sytem LipQ-LipR. The transcription of the lipase operon requires the presence of the alternative sigma factor RpoN (sigma 54) and the transcriptional activator LipR. In P. aeruginosa PAO1, the two-component regulatory system CbrA-CbrB, which controls the utilization of multiple carbon and nitrogen sources, shares more than 80 % of identity with the two-component regulatory system LipQ-LipR of P. alcaligenes. To understand the role of the CbrA-CbrB two-component system in environmental adaptation of strain PAO1, we focus on the regulation of lipase expression. We used translational and transcriptional lipA-lacZ fusions in rpoN, cbrB and hfq mutant strains. In addition, we measured lipase activity in the extracellular medium. The results showed that lipA expression was positively regulated by the sigma factor sigma 54, the response regulator CbrB and also by Hfq. The involvement of Hfq in the positive regulation of lipA suggests that a small regulatory RNA could participate in this regulation.

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Soluble quinone oxidoreductase enzymes of Pseudomonas putida and Pseudomonas aeruginosa: their role in defending against oxidative stressDavid F Ackerley1, AC Matin2 and Laura Green1

1Victoria University of Wellington, New Zealand, 2Stanford University, USA

Most bacteria contain multiple water-soluble quinone-reducing flavoenzymes (quinone oxidoreductases; QOR). Prior to this work, no clear biological benefit for their quinone-reducing capabilities has been demonstrated. ChrR of P. putida is a QOR that has previously been characterised as a chromate reductase. However, we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H2O2. Expression of chrR was induced by H2O2, and levels of ChrR in over-expression, wild type, and knock-out mutant strains correlate with H2O2 tolerance and quenching ability of each strain. The chrR expression level also correlated with intracellular H2O2 levels as measured by flow cytometry using an H2O2-responsive fluorogenic dye. More recently we have been testing whether these novel antioxidant enzymes promote virulence of P. aeruginosa by enabling it to resist host oxidative defences, including the oxidative burst of activated macrophages. The major catalase, KatA, of P. aeruginosa has previously been identified as a virulence factor, highlighting the importance of guarding against host oxidative defences during pathogenesis. However, as KatA has a very high Km for H2O2 it is likely that antioxidant systems with a greater affinity for H2O2

also contribute to virulence. This paper will present our latest findings.


Metabolic impact of organic solvents on solvent-tolerant Pseudomonas putida Lars M. Blank1, Jana Rühl1, Birgitta E. Ebert1, Bruno Bühler1 and Andreas Schmid1

1TU Dortmund, Germany

Besides the classical extremophile conditions high/low pH and high temperatures, environmental niches at which microorganisms are exposed to organic solvents are of interest in such diverse fields as bioremediation and industrial biotechnology. Selected and adapted strains of Pseudomonas putida were reported to tolerate high concentrations of organic solvents and grew in the presence of a second octanol [1], toluene [1], and styrene [2] phase. We are interested in the metabolic impact of these organic solvents, with the latter goal to evaluate if the solvent-tolerant P. putida strains can be used as whole-cell biocatalysts under extreme conditions. Using 13C tracer based metabolic flux analysis, we investigated in solvent-tolerant P. putida strains the interrelationship of butanol or octanol tolerance with energy metabolism and quantified the NAD(P)H regeneration rate in the presence of these toxic solvents. The harsh growth conditions increased the energy demand of the solvent-tolerant P. putida strains. We show that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. This points to a high energy and redox cofactor demand for cell maintenance. Interesting for the application of these organisms for redox biocatalysis, an estimated upper bound for the NAD(P)H regeneration rate available for redox biocatalysis suggests that cofactor availability does not limit biocatalysis under optimized conditions, for example, in the absence of toxic solvent. The results are discussed in the context of the applicability of these extremophiles as hosts for industrial biotechnology. 1. Blank, L. M., Ionidis, G., Ebert, B. E., Buhler, B. & Schmid, A. (2008) Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase, Febs J.2. Park, J. B., Buhler, B., Panke, S., Witholt, B. & Schmid, A. (2007) Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120DeltaC, Biotechnol Bioeng. 98, 1219-1229.


Alanine: its what’s for dinner in the CF lungMegan L Boulette1, Patricia Baynham2, Peter Jorth1, Irena Kukavica-Ibrulj3, Aissa Longoria2, Karla Barrera2, Roger Levesque3 and Marvin Whiteley1

1The University of Texas at Austin, 2St. Edward's University, Austin, TX, 3Laval University, Québec, Canada

Pseudomonas aeruginosa is a Gram negative soil bacterium and opportunistic pathogen. Its chronic association with cystic fibrosis (CF) patients compromises the pulmonary function and longevity of these individuals. In addition, it is also a leading cause of ophthalmic, burn wound, and nosocomial infections, among others. The ability of P. aeruginosa to colonize vastly different environments, often out-competing established organisms including Staphylococcus aureus, is due in part to its propensity to metabolize diverse substrates as sources of carbon and energy. Prior work from our laboratory determined alanine is one of the most abundant carbon sources in CF sputum, the nutritional medium during CF infection, and among those preferentially-consumed by P. aeruginosa. In other organisms, alanine catabolism is mediated by the dadAX operon, which encodes D-alanine dehydrogenase and an alanine racemase and is induced by the transcriptional regulator Lrp. Interestingly, our laboratory also reported that dadAX and lrp are induced by P. aeruginosa during growth in CF sputum and chemically-defined synthetic CF sputum medium (SCFM), as well as in an intraperitoneal model of infection. In this study, we have analyzed the dadAX operon in P. aeruginosa, demonstrating its importance for growth on L- and D- alanine and valine, and describe its regulation by the presence of these amino acids and Lrp. We also discovered the dadA mutant was reduced in its ability to compete with wild type PA14 in SCFM and an in vivo model of infection, and this mutant is defective in its ability to kill S. aureus cultures as well. Our findings suggest that alanine utilization may impact the ability of P. aeruginosa to colonize and compete during the establishment of infection.

P-39

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Topic: Metabolism and Physiology

Mutational analysis of Pseudomonas aeruginosa OxyR to define the regions involved in peroxide resistance and acute virulenceWan-Je Cho1, In-Young Chung1, Bo-Young Lee1, Kyoung-Hee Choi1 and You-Hee Cho1

1Sogang University, Seoul, South Korea

OxyR is known as a hydrogen peroxide (H2O2)-sensing transcriptional activator of katA, katB, aphB, and ahpC responsible for hydrogen peroxide resistance in P. aeruginosa. The oxyR deletion mutant becomes defective in survival on aerobic serial dilution, hypersensitive to H2O2, and impaired in acute virulence in mouse and Drosophila melanogaster infections. To identify the regions of OxyR important in those phenotypes, we carried out site-directed point mutagenesis of the 14 amino acid residues of P. aeruginosa OxyR, based on the amino acid residues known to be implicated in DNA binding, oligomerization, H2O2-sensing, and transcriptional activation of Escherichia coli OxyR. These point mutant alleles of oxyR were introduced into the P. aeruginosa oxyR null mutant at the chromosomal attTn7 and examined for their ability to restore the oxyR mutant phenotypes. Four mutants (C25S, D142A, T129A, and S241A) could complement the aforementioned oxyR mutant phenotypes, whereas S33N, R50A, G96D, G102R, E126K, E228K, and R277H mutants could not fully complement those phenotypes, indicating that the critical involvement of DNA binding, oligomerization and transactivation in OxyR function in vivo, although several residues might be differentially or not implicated in those OxyR functions in P. aeruginosa. Interestingly, the three cysteine mutants (C199S, C208S, and C296S) displayed hypersensitivity to H2O2 and CHP, but only C199S mutant was attenuated in acute virulence, with elevated amount of KatA and AhpC, in contrast to the C208S and C296S mutants, both of which were impaired in the transcriptional activation of katA and katB promoters, indicating that the C296 residue is also involved in the transcriptional activation of OxyR. This dismantling of the OxyR regions implicated in acute virulence and peroxide sensitivity suggests that subtle changes in cytoplasmic redox environment may critically affect the OxyR functioning for the survival within host environments during the infections caused by P. aeruginosa.


Burkholderia pseudomallei and B. thailandensis produce long chain rhamnolipids by the means of two identical rhl operonsEric Déziel1, Danielle Dubeau1, Donald E. Woods2 and François Lépine1

1INRS-Institut Armand-Frappier, Laval, Canada, 2University of Calgary Health Sciences Centre, Canada

Rhamnolipids are surface active molecules composed of rhamnose and beta-hydroxydecanoic acid. These biosurfactants produced by Pseudomonas aeruginosa have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in multicellular behaviours such as biofilm development and swarming motility, and as virulence factors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. We have identified homologous rhlA, rhlB and rhlC biosynthesis genes in B. pseudomallei, an important pathogen, as well as in the genetically resembling non-pathogenic species B. thailandensis. Contrary to P. aeruginosa, these Burkholderia species contain these three genes necessary for rhamnolipid production within a single operon. Furthermore, we found that there are two identical copies of this operon on the second chromosome of these bacteria. Both species are able to produce rhamnolipids distinct from those described for P. aeruginosa, as they are essentially composed of 3-hydroxy fatty acid moieties with longer side chains. Additionally, we found that molecules containing two rhamnose moieties are largely predominant. Separate mutations in both rhlA genes revealed that each copy of the rhl operon is functional, despite the fact that one seems to contribute predominantly to the production compared to the other. Finally, with a double rhlA mutant, which completely lacks rhamnolipid production, we were able to determine that swarming motility of B. thailandensis is dependant on both rhl operons.


Protein-protein and protein-DNA interactions of the DNA polymerase III holoenzyme of Pseudomonas aeruginosaSirine El Houry1, Ute Curth1 and Claus Urbanke1

1Hannover Medical School, Institute for Biophysical Chemistry, Germany

DNA polymerase III is the main enzyme responsible for duplicating DNA during bacterial replication. It's a large protein complex composed of 3 sub-complexes: (1) the core [subunits: alpha, epsilon, theta] contains the polymerase and the proofreading activities, (2) the beta2 sliding clamp [subunit: beta] is a ring-shaped homodimer that tethers the core to its DNA substrate, (3) and the clamp loader [subunits: tau/gamma, delta, delta', chi, psi] is responsible for the opening of the beta2 clamp to load it onto DNA. Other proteins work in coordination with the DNA polymerase III, including the single-stranded DNA binding protein (SSB), which binds to the ssDNA stretches that occur at the replication fork and protects them against nucleases and hairpin formation. It also interacts with the chi subunit of the clamp loader sub-complex. The psi subunit of the clamp loader plays the important role of bridging chi to the rest of the polymerase. Together, chi and psi form a heterodimer. In Pseudomonas aeruginosa, the chi/psi heterodimer has, under low salt conditions, a stimulatory effect on the replication activity of the polymerase, whereas in Escherichia coli, no such effect is observed.Analytical ultracentrifugation was used to analyse protein-protein and protein-DNA interactions in vitro. Studies showed that the binding affinity of the chi/psi heterodimer to SSB is similar in P. aeruginosa and E.coli. Furthermore, P. aeruginosa chi/psi was able to interact with E.coli SSB, and vice versa. These "cross" interactions showed similar binding affinities as well. A "chimeric" chi/psi heterodimer, composed of the chi subunit of E.coli and the psi of P. aeruginosa, was produced, and was shown to bind to either of the SSB proteins. With these findings, in vivo studies of subunit interchange ability between the two species are made possible. On the other hand, it was found that, contrary to the E.coli chi/psi, the P.aeruginosa chi/psi as well as the "chimeric" chi/psi can bind to DNA, and the significance of this interaction is being studied in detail, as it could account for the stimulatory effect of the heterodimer on the replication activity.

P-42

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Topic: Metabolism and Physiology

Role of the RNase R in the turnover of Pseudomonas putida mRNAsPilar Fonseca1, Renata Moreno1 and Fernando Rojo1

1Centro Nacional Biotecnologia CSIC, Madrid, Spain

Pseudomonas putida is a ubiquitous Gram-negative bacterium with a great metabolic versatility and able to colonize different habitats. P. putida is a useful tool in bioremediation, biotechnology and agriculture, and therefore it is important to understand how this microorganism works. In particular, maintaining a proper balance between synthesis and degradation of cellular RNA is essential for attaining adequate levels of gene expression. Degradation of mRNAs in bacteria involves the action of many nucleases. In Escherichia coli, five endonucleases and eight exoribonucleases have been described. The RNase R, a 3 prime to 5 prime exoribonuclease that is very processive with highly structured RNAs, has been shown to be essential for growth at low temperatures in Pseudomonas syringae and in P. putida. In this work, we have analyzed the role of the RNase R in the degradation of mRNAs using a genomic approach. The expression of the RNase R in different conditions has also been analyzed. The results show that the RNase R is very important for the degradation of mRNAs even under conditions in which its absence does not compromise bacterial growth. At 30 degrees, the absence of RNase R leads to increased levels of many mRNAs, and in several cases this is due to an increase in mRNA stability, suggesting that they are direct targets for the RNase R. The absence of RNase R also produces compensatory changes in the RNA decay machinery. We have compared as well the expression pattern of P. putida RNase R under different conditions, concluding that it is different to that of other model bacteria such as E. coli, probably reflecting the different life styles of the microorganisms.

References:- P. Fonseca, R. Moreno & F. Rojo. (2008). Genomic analysis of the role of RNase R in the turnover of Pseudomonas putida mRNAs. J. Bacteriol. 190, 6258-6263.


Spontaneous deletions within a mini-Tn5 transposon of a pcaIJ mutant overcomes lethal knock-out in the benzoate degradation pathwaySarah Frank1, Christoph Ulmer2, Vitor A.P. Martins dos Santos2, Burkhard Tümmler1 and Jens Klockgether1

1Clinical Research Group OE6711, Hannover Medical School, 2Systems and Synthetic Biology Research Group, Helmholtz-Centre for Infection Research (HZI), Germany

The metabolically versatile bacterium Pseudomonas putida is able to degrade a wide variety of pollutants and aromatic compounds and can utilize many of them as a single carbon source. Pseudomonas putida KT2440, a genetically modified lab strain missing the TOL plasmid, only contains the ortho-cleavage branch of the beta-ketoadipate pathway to utilize benzoate and its degradation products. The required enzymes are encoded by three essential gene clusters, the ben (benABCD), cat (catABC) and pca (pcaDIJF) clusters.Screening of a P. putida KT2440 mini-Tn5 transposon library revealed a pcaIJ mutant able to grow on benzoate as a sole carbon source, though a knock-out in these genes was supposed to be lethal. This finding led to the hypothesis that a paralogous operon exists in the P. putida genome that can compensate for the pcaIJ knock-out (Reva et al., 2003).In contrast to the hypothesis, we observed that spontaneous deletions within the mini-Tn5 transposon are responsible for overcoming the knock-out and enabling the mutant to grow on benzoate as a sole carbon source. We cultivated the pcaIJ mutant on M9 medium with benzoate as a sole carbon source. All cultures that grew on benzoate showed a deletion in the transposon sequence. If no spontaneous deletions occurred, the knock-out in the pcaIJ operon was lethal. Sequencing of six independent mutants revealed partial deletions within the same area of the transposon sequence. Analysis of gene expression by RT-PCR of the spontaneous deletion mutants displayed one transcript covering both genes in amounts comparable to the wild type whereas the initial pcaIJ mutant expressed only pcaI.This result demonstrates that bacteria under high selective pressure can implement rescue mechanisms to overcome lethal mutations and can be seen as an example of evolutionary adaptation. In this special case the pcaIJ mutant is able to modulate even a mini-Tn5 transposon insertion that is described to induce very stable mutant phenotypes (de Lorenzo and Timmis, 1994).


A new vision of the metabolism of P. putidaMaría Eugenia Guazzaroni1, Manuel Ferrer1 and Florencio Pazos2

1Institute of Catalysis, CSIC, 2Centro Nacional de Biotecnología, CSIC, madrid, Spain

Objective: To study the catabolic behaviour of P. putida under solvent-stress.Pseudomonas putida DOT-T1E has the huge capacity to resist and grow in the presence of high concentrations of organic solvents, such as toluene. The main mechanism underlying this type of resistance has been found in the action of three RND efflux pumps, called TtgABC, TtgDEF and TtgGHI, which extrude organic solvents and other toxic compounds from the cells (Ramos et al., 1995, Ramos et al., 2002). This ability has generated a considerable interest in exploiting this bacterium as a cell factory in a diverse range of biotechnological applications, including bioremediation of contaminated sites and biocatalysis for the production of fine chemicals (Schmid et al., 2001; Rojas et al., 2004). Although to date several studies at proteomic (Segura et al., 2005), and fluxomic level (del Castillo et al., 2007; del Castillo and Ramos, 2007) have been carried out with this strain, there is not experimental information of comprehensive features of its metabolic capacity. An analysis of the metabolism of P. putida DOT-T1E grown with and without toluene was carried out. Here, we present new information of how this bacterium can tolerate organic solvents and suggest new windows of catabolic activities under solvent-stress.del Castillo, T., and Ramos, J.L. (2007) J Bacteriol 189: 6602-6610.del Castillo, T., Ramos, J.L., Rodriguez-Herva, J.J., Fuhrer, T., Sauer, U., and Duque, E. (2007) J Bacteriol 189: 5142-5152.Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., Ramos-Gonzalez, M.I., Rojas, A., Teran, W., and Segura, A. (2002) Annu Rev Microbiol 56: 743-768.Ramos, J.L., Duque, E., Huertas, M.J., and Haidour, A. (1995) J Bacteriol 177: 3911-3916.Rojas, A., Duque, E., Schmid, A., Hurtado, A., Ramos, J.L., and Segura, A. (2004) Appl Environ Microbiol 70: 3637-3643.Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., and Witholt, B. (2001) Nature 409: 258-268.Segura, A., Godoy, P., van Dillewijn, P., Hurtado, A., Arroyo, N., Santacruz, S., and Ramos, J.L. (2005) J Bacteriol 187: 5937-5945.

P-45 Removed by request of the author before online publication.

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MucR is involved in regulation of alginate biosynthesis in Pseudomonas aeruginosaIain D Hay1, Uwe Remminghorst2 and Bernd H. A. Rehm2

1Massey University , 2Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand

Alginate is an extracellular polysaccharide of clinical importance produced by Pseudomonas aeruginosa. The emergence of mucoid (alginate overproducing) forms of the bacteria is recognised as a poor prognosis indicator among patients susceptible to infection. It is thought that the production of copious amounts of this extracellular matrix can be advantageous in protecting the bacteria from environmental stresses and host defences. Recently a novel regulator of alginate biosynthesis, MucR, has been identified. MucR is a membrane anchored digunulate cyclase that is thought to positively regulate alginate biosynthesis through producing bis(3’-5’)-cyclic dimeric GMP (c-di-GMP) which is subsequently bound by the Alg44. Alg44 and its c-di-GMP binding domain have previously been shown to be essential for alginate biosynthesis. Deletion of the mucR gene in a mucoid background resulted in a marked reduction in alginate production, and over expression of mucR led to an increase in alginate production beyond that of the wild type, as well as the emergence of several phenotypes characteristics of increased c-di-GMP levels. Here the membrane topology of MucR was probed and the functional role of both the GGDEF and EAL domains was investigated with respect to their requirement for alginate biosynthesis. Additionally the role of MucR in the transcriptional regulation of the alginate operon was addressed.


The major catalase (katA) gene of Pseudomonas aeruginosa PA14 is under both positive and negative control by the global transactivator OxyR in response to hydrogen peroxideYun-Jeong Heo1, In-Young Chung1, Kyoung-Hee Choi1, Wan-Je Cho1, Jin-Won Lee2, Daniel Hassett3 and You-Hee Cho1

1Sogang University, Seoul, South Korea, 2Hanyang University, South Korea 3University of Cincinnati College of Medicine, USA

Microbes have the capacity to rapidly adapt to peroxide stress. In particular, the peroxide-sensing transcriptional regulator OxyR plays a critical role in the regulatory networks governing such responses. The adaptive response to hydrogen peroxide (H2O2) in Pseudomonas aeruginosa involves the major catalase, KatA and OxyR. However, neither the molecular basis nor the relationship between the aforementioned proteins has been established. Here, we demonstrate that the transcriptional activation of the katA promoter (katAp) in response to H2O2 was abrogated in the P. aeruginosa PA14 oxyR null mutant. Promoter deletion analyses revealed that H2O2-mediated induction was dependent on a region of DNA -76 to -36 upstream of the H2O2-responsive transcriptional start site. This region harbored the potential operator sites (ORE, OxyR-responsive element) of the Escherichia coli OxyR binding consensus. Deletion of the entire ORE not only abolished H2O2-mediated induction, but also elevated the basal transcription, suggesting the involvement of OxyR and ORE in both transcriptional activation and repression. OxyR bound to the ORE both in vivo and in vitro, demonstrating that OxyR directly regulates the katAp. The predominant species of oxidized OxyR upon H2O2 exposure was sulfonic acid (-SO3H) form at the conserved peroxidatic cysteine (C199) in P. aeruginosa. The uninduced transcription of katAp was also highly elevated in an oxyR mutant for C199 (C199S), but not in an oxyR mutant for C208 (C208S). In both mutants, however, katAp transcription was partially induced by H2O2 treatment, unlike in the oxyR null mutant; the H2O2-induction was delayed in the C199S mutant and lower in the C208S mutant. Taken together, our results suggest that P. aeruginosa OxyR is a bona fide transcriptional regulator of the katA gene, sensing H2O2 based on the conserved cysteines. Furthermore, the unmodified Cys 199 thiol is required for the repression of the katA transcription and the Cys 208 thiol oxidation is required for the transcriptional activation.


Identification of genes implicated in glucose-dependent cell lysis of Pseudomonas putidaRita Hõrak1, Marta Putrinš1 and Külliki Püvi1

1University of Tartu, Estonia

Successful survival of bacteria in ever-changing conditions depends on their ability to monitor the environment and to translate external signals into adaptive responses. Two-component signalling pathways are one of the most prevalent means by which bacteria transduce information from outside to inside the cell in order to adjust their behaviour according to the new situation. We are aiming to uncover the physiological role of signal transduction pathway ColRS in Pseudomonas putida. Our recent studies revealed that the deficiency of ColRS system results in lowered phenol tolerance of P. putida, hindrance of transposition of Tn4652 and lysis of a subpopulation of glucose-grown bacteria (Horak et al., 2004; Kivistik et al., 2006; Putrins et al., 2008). The last phenotype of the colR mutant was observed to be accompanied with increased aggregation and Congo Red staining of bacteria. The Congo Red binding phenotype was used in current study to search for mutants that either can suppress glucose-induced cell lysis of the colR-deficient strain or, in contrary, can evoke the lysis of wild type P. putida. Screening of transposon insertion libraries of both strains on glucose-Congo Red plates revealed a set of non-lysing mutants from the colR-deficient strain as well as lysis-prone mutants from the wild-type. Analysis of transposon derivatives revealed that the non-lysing phenotype of colR-deficient strain occurred due to disruption of different genes, such as genes of glucose transport, outer membrane protein OprF, chaperone SecB etc. Interestingly, disruption of secB, which is involved in protein secretion, restored also the transposition of Tn4652. This indicates that protein composition of the membrane of colR mutant could be unbalanced and that defect in turn affects both glucose-dependent cell lysis and mutagenic processes. Analysis of lysis-prone transposon derivatives of the wild type strain disclosed besides colR and colS several other genes whose disruption caused glucose-dependent cell lysis of P. putida. Interestingly, one of those mutants behaved very similar to the colR-deficient strain revealing phenotypes of lowered phenol tolerance and hindrance of transposition of Tn4652. This gene coding for a hypothetical protein of the TonB family, as well as some other genes identified by transposon library screens are currently under investigation to clarify their potential interaction with the ColRS signalling pathway.


Metabolic impact on virulence in Pseudomonas aeruginosaSonja Horatzek1, Eliane Frimmersdorf2, Lutz Wiehlmann1, Dietmar Schomburg2 and Burkhard Tümmler1

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1Clinical Research Group OE6711, Hannover Medical School, 2Institute of Bioinformatics & Biochemistry, TU Braunschweig, Germany

Quorum sensing (QS) is a key regulatory system that allows bacterial populations to coordinate cell-density-dependent gene expression. Based on cell-to-cell signalling achieved by chemical signals, this process controls hundreds of genes in P. aeruginosa, many of which encode extracellular virulence factors. This opportunistic human pathogen possesses a broad array of these pathogenicity-associated exoproducts, including toxins and hydrolytic enzymes, which are involved in local tissue damage, dissemination and pathogenesis of acute infections.In a STM screen of P. aeruginosa strain TBCF10839, a clinical isolate from a Cystic Fibrosis patient, we identified several transposon mutants, deficient in QS and thus unable to produce QS-dependent virulence factors under minimal media conditions. Interestingly, neither of the candidate genes is directly involved in QS or virulence. In fact, the different gene functions each can be associated with the central energy metabolism which indicates a strong impact of metabolism on QS and virulence in P. aeruginosa of yet unknown nature.In order to identify metabolic pathways important for the regulation of QS-associated virulence comparative metabolome analysis was performed. The results support the hypothesis of a central regulatory switch that, strongly dependent on the cell's metabolic and physiological condition, controls energy fluxes towards QS-associated processes.QS deficiency in the knock-out mutants can be overcome by supplementation with different carbon and energy sources which provides experimental evidence for the tight connection between the bacterial cell's metabolic state and the expression of QS-dependent virulence factors.These findings are to be considered in the application of knock-out mutants in in vivo models, since the nutritional environment in the host may significantly alter the virulence competence of these mutants which conflicts with their in vitro phenotype.


Evolution of Pseudomonas aeruginosa during chronic lung infectionHolly K. Huse1, Taejoon Kwon2 and Marvin Whiteley2

1University of Texas at Austin, 2University of Texas at Austin 2506 Speedway NMS 3.254 Austin, TX 78712, USA

The Gram-negative bacterium Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with the heritable disease Cystic Fibrosis (CF). P. aeruginosa chronically colonizes the lungs of CF patients, where it reaches high cell densities and produces a battery of virulence factors. Upon infection, a single strain of P. aeruginosa can colonize a patient's lungs throughout his or her lifetime. To understand the evolution of P. aeruginosa during chronic lung infection, we conducted both genotypic and phenotypic analyses on clinical isogenic strains obtained from the lungs of three different CF patients. These strains were isolated over a period of approximately ten years and possess phenotypes that are commonly observed in isolates from the CF lung, such as classic, dwarf, and mucoid. Microarray analyses were carried out on isolates grown in a chemically defined medium that mimics the nutritional environment of the CF lung, synthetic CF sputum medium (SCFM). Interestingly, hierarchical clustering by differential gene expression demonstrated that most strains cluster together by patient rather than by phenotype. In addition, phenotypic analyses on the first and last patient isolates have demonstrated that later isogenic strains lose the ability to grow on small acids. We are currently sequencing the genomes of the first isolates from each patient to determine genotypic differences amongst strains. These studies give insight on the evolution of a common human pathogen during chronic lung infection.


Characterization of two Pseudomonas aeruginosa OhrR homologs in response to organic hydroperoxidesMin-Gyeong Kang1, Seol-Hee Kim1 and You-Hee Cho1


OhrR is known as a novel organic peroxide-inducible transcriptional repressor, which can be classified into two subgroups based on the number of cysteine residues required to sense and respond to organic hydroperoxides. We indentified that two OhrR homologs (OhrR, PA14_27230 and OhrR2, PA14_27530) are present in the genome of Pseudomonas aeruginosa PA14, both of which belong to multi-cysteine subgroups, with three cysteines (C9, C19 and C121) and two cysteines (C24 and C134) for OhrR and OhrR2, respectively. Based on Northern blot analyses using ohrR, ohrR2, and ohrRR2 null mutants, we found that the transcription of the monocistronic ohr gene was increased in ohrR and ohrRR2, but not affected at all in ohrR2 mutants, and both ohrR and ohrRR2 mutants showed increased resistance to CHP and tBHP as well as to hydrogen peroxide than ohrR2 mutants. Mutational analysis of each cysteine residues of OhrR revealed that one single cysteine (C19) among the three cysteines was sufficient to respond to cumene hydroperoxide (CHP) and t-butyl hydroperoxide (tBHP). OhrR could bind to the promoter upstream region of the ohr gene in only at its reduced state. OhrR could form intersubunit disulfide bond, resulting in the loss of the DNA binding ability, when it was oxidized by CHP. These results suggest that OhrR is the OhrR ortholog in response to organic hydroperoxides, whereas the physiological role of OhrR2 remains elusive.


Novel antimicrobial compounds produced by rhizosphere PseudomonasWen Li1, Guanlin Xie2 and René De Mot3

1Centre of Microbial and Plant Genetics, 2Institute of Biotechnology, Zhejiang University,310029 Hangzhou,China, 3Centre of Microbial and Plant Genetics, K.U.Leuven, B-3001 Heverlee, Belgium

Pseudomonas strains produce a variety of antimicrobial molecules contributing to biocontrol of phytopathogenic fungi, but much less is known about compounds mediating antagonism of other plant-associated bacteria. Our work focuses on the characterization of such antimicrobial compounds from two Pseudomonas rhizosphere isolates.The banana rhizosphere isolate Pseudomonas sp. BW11M1 produces a lectin-like bacteriocin killing several (phytopathogenic) Pseudomonas (Parret et al. 2003, Journal of Bacteriology 185: 897). Its anti-Xanthomonas activity is not bacteriocin-mediated but involves three non-ribosomal peptide synthetase (NRPS) genes organized in two separate clusters (approximately 46 kb). The secondary metabolite involved (presumably a lipopeptide) also inhibits some phytopathogenic fungi (Botrytis cinerea, Rhizoctonia solani). In addition, BW11M1 appears to produce a third as yet uncharacterized antimicrobial compound with anti-Xanthomonas activity.

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The rice rhizosphere isolate Pseudomonas sp. RW10S1 displays antagonistic activity against several Pseudomonas species. The GacS/GacA two-component system controls expression of this anti-Pseudomonas activity. The biosynthetic gene cluster (15.7 kb) with an unprecedented combination of genes points to involvement of salicylic acid biosynthesis, amino acid incorporation and fatty acid metabolism, along with some as yet unidentified activities. Currently, the structure of this novel antibiotic is being determined by mass spectrometry and NMR.


Characterization of a potential heme regulator in Pseudomonas aeruginosaJuan Carlos Lorenzo Fajardo1, Katja Weichbrodt2, Andrea Harms2, Katalin Barkovits1, Britta Schubert1 and Nicole Frankenberg-Dinkel1

1Ruhr-University Bochum, Physiology of Microorganisms, 2 Technical University Braunschweig, Institute for Microbiology, Germany

Sensing and adaptation to external environmental changes is crucial for all microorganisms. However, maintaining internal homeostasis of important but potentially cytotoxic compounds like heme is equally important. One way to sense internal signals is the use of LysR-type transcriptional regulators (LTTRs) which combine sensing and transcriptional regulation of multiple genes via a co-inducer binding domain and a helix-turn-helix motif, respectively. Here we describe the characterization of such an LTTR from Pseudomonas aeruginosa which was named HemR due to its high homology to the heme dependent regulator HbrL from Rhodobacter capsulatus. Spectroscopic experiments, using recombinantly produced HemR showed that neither heme nor the heme degradation product biliverdin-IXa is able to bind to HemR. However, HemR was found to be involved in maintaining heme homeostasis by repressing hemA, encoding the first, rate limiting enzyme of heme biosynthesis and activating bphO, a heme degrading heme oxygenase. Furthermore, our results point towards a discrimination between internal and external heme sources. In contrast to many other LTTRs, the expression of hemR is not autoregulated but a strong repression of the adjacent gene PA2929, annotated as a putative LysE-type exporter, was observed. The potential involvement of this exporter in heme homeostasis will be discussed.


Structural Insight into the catalytic mechanism of 4-methylmuconolactone methylisomeraseMacarena A Marin1, Björn Klink1 and Dietmar Pieper1

1Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany

The 3-oxoadipate pathway is central for bacterial metabolism of aromatics. However, this pathway is usually not suited for degradation of methylaromatics, which are typically mineralized via extradiol cleavage. In both Cupriavidus necator JMP134 and Pseudomonas reinekei MT1 a novel variant of the 3-oxoadipate pathway was identified, with a 4-methylmuconolactone methyl isomerase (MLMI) as key enzyme. MLMI catalyzes the transformation of 4-methylmuconolactone (4ML) to 3-methylmuconolactone (3ML). MLMI belongs to the MmlI protein superfamily, where only a few members are known, and the enzyme mechanism has not been clarified yet. Here we present the crystal structures of the MLMI from Pseudomonas reinekei MT1 in its apo state at 1.5 Å and in complex with its product (3ML) at 1.5 Å. Analysis of the binding site indicated a tyrosine (Tyr-54) and two histidines (His-41 and His-67) as the most probable catalytic residues. Site directed mutagenesis of His-41 and His-67 to alanine and Tyr-54 to phenylalanine decreased activity. The H67A derivative could be crystallized in complex with the substrate (4ML) at 1.8 Å, allowing the identification of its orientation in the active site. Based on these results, a novel catalytic mechanism is proposed where His-41, His-67 and Tyr-54 play a key role in the catalysis.


The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analysesRenata Moreno1, Montserrat Martínez-Gomariz2, Luis Yuste3, Concha Gil4 and Fernando Rojo3

1Centro Nacional de Biotecnología, CSIC, 2Unidad de Proteómica-Parque Científico de Madrid, Universidad Complutense, 28040-Madrid, Spain, 3Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049-Madrid, Spain, 4Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain

The Crc protein is a global regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in pseudomonads when other preferred substrates are present. Crc binds to the 5-prime end of target mRNAs, inhibiting translation initiation. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in P. putida KT2442 cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for Pro, Ala, Glu, Gln and His. These amino acids are good carbon sources for P. putida. In the case of Arg, Lys, Asp and Asn, which can be assimilated through several pathways, Crc favoured one particular route, inhibiting other alternatives. Finally, Crc inhibited genes needed to assimilate Val, Ile, Leu, Tyr, Phe, Thr, Gly and Ser, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.

Moreno, R., Ruiz-Manzano, A., Yuste, L., Rojo, F. (2007). The P. putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. Mol. Microbiol. 64:665.Moreno, R., Rojo, F. (2008). The target for the P. putida Crc global regulator at the benzoate degradation pathway is the BenR transcriptional regulator. J. Bacteriol., 190:1539.Moreno, R., Martínez-Gomariz, M., Yuste, L., Gil, C., Rojo, F. (2009). The P. putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analyses. Proteomics, in press.


Siderophores of Pseudomonas syringae: synthesis and biological significanceJeremy Owen1 and David Ackerley1

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1Victoria University of Wellington, New Zealand

Pseudomonas syringae pv. phaseolicola 1448a, like many pseudomonads, is capable of occupying diverse biological niches. It is also the causative agent of bean halo blight. Under conditions of iron starvation P. syringae relies on siderophores for the acquisition of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent molecule with high affinity for iron. Pyoverdine is synthesised from amino acid precursors by Non-Ribosomal Peptide Synthases (NRPS). The secondary siderophore of P. syringae, achromobactin, has a lower affinity for iron and its contribution to iron uptake is usually masked by pyoverdine under conditions of iron starvation. We have produced in-frame deletions of each of the five NRPS genes thought to govern pyoverdine biosynthesis in P. syringae and the role of these genes in pyoverdine biosynthesis has been confirmed. We have also created a mutant strain lacking the ability to produce achromobactin as well as a strain lacking both achromobactin and pyoverdine. The phenotype of P. syringae lacking pyoverdine, achromobactin and both siderophores has been characterised with regards to iron uptake, pathogenesis of beans and growth in iron limited conditions. Our results suggest that pyoverdine is more important for survival during iron starvation than achromobactin. Also reported here are techniques for the purification and characterisation of NRPS adenylation domains that were derived during attempts to characterise the substrate specificity of the pyoverdine NRPS of P. syringae. This research sheds new light on the biosynthesis and genomics of siderophores in P. syringae as well as their physiological roles.


Metabolome analysis of P. aeruginosa clinical isolatesAnya Pelnikevich1, Elza Rakhimova1, Sonja Horatzek1, Burkhard Tümmler1, Dietmar Schomburg2 and Lutz Wiehlmann1

1Clinical Research Group OE6711, Hannover Medical School, 2Institute of Bioinformatics & Biochemistry, TU Braunschweig, Germany

Pseudomonas aeruginosa is a ubiquitous environmental soil bacterium and an opportunistic pathogen of humans, animals and plants. It causes chronic infections in patients with cystic fibrosis (CF), chronic obstructive pulmonary disease and bronchiectasis. P. aeruginosa isolate TBCF10839 is a highly virulent strain of P. aeruginosa, which belongs to a major clone in the P. aeruginosa population. It is a pilin-deficient strain that produces large amounts of alginate and survives in polymorphonuclear leukocytes. Being a strong producer of virulence effector proteins, it causes substantial airway pathology in mice after intratracheal instillation. Transcriptome and proteome analyses indicated that TBCF10839 orchestrates many more metabolic and signalling pathways upon exposure to inanimate and animate stressors than the strain PAO1. The aims of this work were to initially unravel how the virulence factor production depends on metabolic pathways and the metabolic state of the organism. And secondly to understand how does selective carbon sources control and modulate the activation of specific virulence programs of the P. aeruginosa.In our work we used a set of mutants which have knock-outs in genes of the core metabolism (Entner-Doudoroff pathway, citric acid cycle and redox status of the cell). All of them were shown to be impaired in virulence in mice infection experiments.The metabolomes of these mutants grown under conditions mimicking the environment in patient lungs during the infection were compared by Gas Chromatography - Mass Spectroscopy (GC-MS) in different growth phases. This analysis covers the identification of most intracellular and extracellular metabolites with molecular masses lower than 1000 Da. By comparing the metabolomes of the mutants within one function class and with the wild type, it is possible to identify similarities in the metabolic changes and to identify those alterations that are specific for the growth under infection conditions. The combined metabolome and transcriptome data can be used to estimate the major metabolic fluxes under the conditions resembling the human lung infection process.


Comparison of the membrane proteomic profiles of rhlA and rhlB deletion mutants in Pseudomonas aeruginosa.Bianca L Price1 and Martin Welch1

1University of Cambridge, UK

Rhamnolipids are surfactants produced by Pseudomonas aeruginosa (PAO1). They consist of one or two rhamnose head groups attached to a fatty acid tail, referred to as mono- or di-rhamnolipid respectively. These surfactants have a variety of suggested functions including development and maintenance of biofilm structure, regulation of swarming motility and uptake of hydrophobic substrates. They also have bactericidal properties and are involved in virulence. The PAO1 operon responsible for rhamnolipid biosynthesis consists of rhlA and rhlB. RhlA synthesizes the hydroxyalkanoic acid (HAA) fatty acid tail moiety of rhamnolipids to which RhlB adds a rhamnose group to form mono-rhamnolipid. In order to further characterise the roles of HAAs and rhamnolipids in PAO1 cellular physiology, membrane proteins were harvested from a rhlA mutant, as well as from a rhlB mutant, and the proteomes were profiled using Spectral Ion Counting. Changes in the membrane proteomic profiles mapped to several operons, most notably those responsible for the production of siderophores, pyocyanin and protein secretion machinery. These changes were validated with subsequent phenotypic assays linking the absence of rhamnolipids, or the absence of HAAs and rhamnolipids, with iron uptake, pyocyanin production, swarming motility and biofilm formation. These data provide insights into rhamnolipid secretion, as well as insights into the role these surfactants play in the physiology of P. aeruginosa.


Glucose-dependent lysis of Pseudomonas putida deficient in ColRS two-component system Marta Putrinš1, Heili Ilves1, Maia Kivisaar1 and Rita Hõrak1

1University of Tartu, Estonia

Autolysis of subpopulation of bacteria has been described as a part of different processes like sporulation, biofilm development and stress response. Despite recent advances in this field, understanding about signalling pathways regulating the death decisions in bacteria is far from complete.The two-component system ColRS is well conserved in pseudomonads, but so far its role has been shown only in some unrelated processes. First, it was reported that a colS-deficient P. fluorescens was unable to compete with wild-type bacteria in plant root colonization (Dekkers et. al., 1998). Additionally, we have shown that the ColRS system is implicated in mutagenesis and phenol tolerance in P. putida (Horak et. al., 2004; Kivistik et. al., 2006).

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Our recent results revealed that Pseudomonas putida deficient in ColRS system experiences serious carbon source-specific stress that leads to the lysis of a subpopulation of bacteria growing on solid glucose medium (Putrins et. al., 2008). Importantly, cell lysis of a colR mutant became evident only on solid glucose medium, being not detectable even on gluconate that is degraded through the Entner-Doudoroff pathway like glucose. Therefore, we consider that glucose itself and/or its degradation through glucokinase route should be a source of stress signal that leads to cell lysis of the colR mutant. Interestingly, inactivation of oprB1 encoding a glucose porin eliminated the cell lysis of a colR mutant. We have also constructed an OprB1 overexpression strain that does not enhance cell lysis. This ruled out the possibility that the presence of a high amount of OprB1 in the outer membrane of the glucose-grown colR mutant may somehow destabilize intrinsically fragile membrane of colR-deficient cells leading to lysis. In addition, disruption of genes coding for a glucose transport system also eliminated the cell lysis of a colR mutant indicating that glucose influx could be responsible for membrane stress in the absence of the ColRS system. To further unravel the relationship between the ColRS system and the glucose metabolism, experiments with several glucose catabolism-deficient strains as well as measurements of activities of different Entner-Doudoroff pathway enzymes have been undertaken. Our preliminary results suggest that both glucose transport as well as glucose metabolism through the glucokinase route are implicated in cell lysis of ColRS system-deficient P. putida.


Functional assignment of atuRABCDEFGH genes to specific functions of the acyclic terpene utilization (Atu) pathway in P. aeruginosaNadine Randel1, Karin Förster-Fromme2 and Dieter Jendrossek2

1Universität Stuttgart, 2Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

The catabolic pathway of low molecular compounds with methyl-branched carbon skeletons is poorly understood. Citronellol and related acyclic monoterpenes are naturally occurring compounds in plants and are commonly used as insect repellents, in the cosmetic and food industry. Citronellol can be utilized as a single source of carbon and energy only by P. citronellolis and a few related pseudomonads such as P. aeruginosa. Recently, we identified the atuRABCDEFGH gene cluster of P. aeruginosa and found that it is essential for acyclic terpene utilization [1].The Atu pathway consists of seven enzymatic steps. The atu gene cluster harbours eight genes encoding seven proteins (geranyl-CoA carboxylase consists of two subunits). Six of the seven Atu proteins (except AtuH) are specifically expressed in acyclic terpene-grown cells of P. aeruginosa. Therefore, it is possible that each of the atu genes contributes to one of the individual steps. Putative functions of the atu gene products are based on (i) verified biochemical function of expressed genes (atuC /atuF, atuD) [2, 3] or (ii) high amino acid similarity values to proteins with known functions (atuB, atuE, atuG, atuH). No function, however, could be addressed to AtuA so far. We speculate that AtuA could be a 3-hydroxy-3-iso-hexenyl-glutaryl-CoA acetate lyase (HHG lyase) - the last step of the Atu pathway - as this is the only enzyme whose function is not covered by any of the other Atu proteins. In an attempt to verify this assumption we started to reconstruct the pathway in vitro with purified selected Atu proteins. As an alternative approach the substrate of HHG lyase is chemically synthesised to be used a substrate for purified recombinant AtuA. [1] Foerster-Fromme, K. et al. (2006) AEM 72, 4819-4828.[2] Foerster-Fromme, K. et al. (2008). Microbiology 154, 789-796.[3] Aguilar, J.A. et al. (2008). J Bacteriol 190, 4888-4893.


Characterization of Pseudomonas aeruginosa isolates from urinary tract infectionsNathalie Rosner1, Katrin Zapf2, Lothar Jänsch3, Dietmar Schomburg4, Max Schobert1 and Petra Tielen1

1Technische Universität Braunschweig, Institute of Microbiology, Spielmannstraße 7, 38106 Braunschweig, Germany, 2Technische Universität Braunschweig, Institute of Biochemical Engineering, Gaußstraße 17, 38106 Braunschweig, Germany, 3Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, 4Technische Universität Braunschweig, Institute of Bioinformatics and Biochemistry, Langer Kamp 19B, 38106 Braunschweig, Germany

Pseudomonas aeruginosa is an opportunistic human pathogen which is isolated from diverse aquatic and soil habitats. It causes severe acute and chronic nosocomial infections in immunodeficient patients as lung infections, wound infections or urinary tract infections. In patients with urethral catheters, P. aeruginosa has been described in 35 per cent of the cases as the common strain in complicated urinary tract infections. These strains often show a high intrinsic antibiotic resistance which complicates the medical treatment. We developed an in vitro model system with an artificial urine medium (AUM) as nutrient source, which simulates the conditions during acute urinary tract infection. P. aeruginosa was grown under aerobic and anaerobic conditions in colony biofilms. For the comparison of the nutrient supply, 10fold diluted LB medium served as reference. Comprehensive proteome, metabolome and transcriptome analyses were used to study the specific physiology of P. aeruginosa PAO1 under the chosen conditions. We found an increased level of proteins and transcripts of tricarboxylic acid cycle components as well as a highly induced phenazine biosynthesis under urinary tract like conditions. Moreover, we investigated a catheter biofilm model to simulate the physiology of catheterized patients and started with proteome analysis via 2D gel electrophoresis. Comparing the data of the "omic" techniques using the databases PRODORIC and SYSTOMONAS, we detected adaptations of P. aeruginosa playing a role in urinary tract infections due to antibiotic tolerances and specific biofilm phenotypes.


The two component system TodS/TodT of Pseudomonas putida DOT-T1E: complex domain arrangement and novel mechanismHortencia Silva-Jiménez1, Andreas Busch2, Jesús Lacal2, María-Eugenia Guazzaroni2, Tino Krell2 and Juan Luis Ramos2

1Estación Experimental del Zaidín, 2Estación Experimental del Zaidín, CSIC, c/Profesor Albareda No. 1, Spain

Objective: Determination of the molecular mechanism of the two component system TodS/TodT of Pseudomonas putida DOT-T1E.The two-component system TodS/TodT controls the expression of the tod (toluene dioxygenase) operon in Pseudomonas putida for metabolism of toluene, benzene and ethylbenzene. The sensor kinase TodS has an unusually complex domain arrangement: it contains two modules each containing a PAS-type sensor domain and an autokinase domain. Both modules are separated in sequence by a response regulator receiver domain.

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Although the TOD pathway has only three substrates the activity of the TodS/TodT system is regulated by a variety of monocyclic aromatic compounds (1), which can be classified as agonists, which bind to TodS, modulate its autophosphorylation and active transcription in vivo, and antagonists, which bind to TodS but do not modulate its activity nor increase gene expression (2). Agonists and antagonists compete for the same site at the N-terminal PAS domain. Binding of agonists stimulated the autophosphorylation of the N-terminal kinase domain, which is followed by an intra-TodS phosphoryltransfer involving the receiver domain and the C-terminal autokinase domain prior to phosphoryltransfer to TodT (3). TodT binds to three different sites at its target promoter PtodX in a cooperative and hierarchical manner. ITC studies reveal that the recombinant DNA-binding domain of TodT has similar binding characteristics as compared to the full-length protein but its binding does not translate into promoter activation (4). Atomic force microscopy studies show that the N-terminal domain of TodT is essential for the formation of DNA-loop which appears to be required for efficient transcription initiation (5). This is the first example of a multi-step phosphorelay involving two autokinase domains. The physiological role of this complex mechanism remains unclear but it is hypothesized that TodS recognizes multiple signals which contribute to the final transcriptional output (6).References: 1. Lacal et al. (2006) Proc. Natl. Acad. Sci. USA 103: 81912. Busch et al. (2007) Proc. Natl. Acad. Sci. USA 104: 137743. Busch et al. (2009) J. Biol. Chem., in press4. Lacal et al. (2008) J. Mol. Biol. 376: 3255. Lacal et al. (2008) J. Mol. Biol. 384: 1037 6. Krell et al. (2009) Environ. Microbiol.Reports, in press


Stringent response and the ribosome modulation factor in pseudomonadsAnnika Steen1, Nelli Bös1, Mathias Müsken2, Susanne Häußler2, Maurice Scheer3 and Max Schobert1

1Technische Universität Braunschweig, Institute of Microbiology, Spielmannstr.7, 38106 Braunschweig, 2Chronic Pseudomonas Infection Research Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, 3Bioinformatics and Biochemistry, Technische Universität Braunschweig, Langer Kamp 19b, 38106 Braunschweig, Germany

The bacterial stringent response is a cellular adaptation to nutrient limitation, such as amino acid or carbon starvation, mediated by the guanosine nucleotide ppGpp. In Pseudomonas aeruginosa and Pseudomonas putida, the proteins RelA and SpoT control the cellular level of ppGpp. In order to investigate the stringent response, we constructed relA spoT double mutants of the opportunistic pathogen P. aeruginosa and its non-pathogenic relative P. putida. We monitored the behaviour of both mutants under various stress conditions and determined conditions suitable for induction of a RelA- or SpoT-dependent stringent response. Microarray analysis was used to identify promoters, which are under control of the stringent response in both bacteria. Furthermore, we carried out an extensive phenotypic characterization of the relA spoT mutant strains.The rmf gene, encoding the ribosome modulation factor, was found upregulated via the stringent response in both pseudomonads. In Escherichia coli, the Rmf protein was shown to mediate the dimerization of 70S ribosomes, leading to an increased tolerance under various stress conditions. Characterization of rmf mutant strains indicated slightly differential roles for the ribosome modulation factor in Pseudomonas. Moreover, the postulated binding site of the Rmf protein on the 30S ribosome subunit overlaps with the target sites of various antibiotics. This suggests that the ribosome modulation factor might contribute to an increased tolerance to ribosome-targeted antibiotics. Interestingly, transcription of the rmf gene was found highly upregulated under conditions in which P. aeruginosa PAO1 displays an increased tolerance to antibiotics such as biofilm growth. We found that the PAO1 rmf mutant strain showed reduced survival rates in mature biofilms incubated on the aminoglycoside tobramycin in comparison to the wild type strain. These results indicate an important role of the ribosome modulation factor in resistance to ribosome-targeted antibiotics.


Three pseudomonads respond differently to harbouring a catabolic plasmidYurika Takahashi1, Masaki Shintani1, Hirofumi Hara2, Koji Kadota3, Hiromi Nishida3, Hisakazu Yamane1 and Hideaki Nojiri1

1Biotechnology Research Center, The University of Tokyo, Tokyo, JAPAN, 2Department of Biomedical Engineering, Okayama University of Science, Okayama, JAPAN, 3Professional Program for Agricultural Bioinformatics, The University of Tokyo, Tokyo, JAPAN

Genus Pseudomonas bacteria usually possess plasmids, which provide their host with various abilities, such as degradative capacity of recalcitrant compounds, resistance to antibiotics and heavy metals, or pathogenicities. The Carbazole-degradative plasmid pCAR1, belonging to the incompatibility (Inc) P-7 group, has transferability to other Pseudomonas and Stenotrophomonas bacteria (Shintani et al., Appl. Environ. Microbiol., 72, 3206-3216, 2006; Shintani et al., Biotechnol. Lett. 30, 117-122, 2008) whose entire nucleotide sequence has already been determined (Maeda et al., J. Mol. Biol., 326, 21-33, 2003; Takahashi et al., Biosci. Biotechnol. Biochem., in press). Although there are many reports that a plasmid affects the physiology of its host, such as growth rate or metabolic capacity, it is still unclear how the changes occurred and differed according to host strains. To address this simple but prime issue, we compared the response(s) of three Pseudomonas bacteria: P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1, to harbouring pCAR1.First, we compared the phenotypes of the pCAR1-free or pCAR1-containing strains. The differences of growth rates between Pf0-1 and Pf0-1(pCAR1) were larger than the others in succinate-supplemented mineral medium (Shintani et al., submitted). It was also observed that motility of the tree strains on 0.3% agar plate was reduced by the carriage of pCAR1.Second, transcriptome analyses of host chromosomes were performed using high-resolution tilling arrays at early log phase on succinate culture. Among many genes differently transcribed by the carriage of pCAR1 (121 in KT2440, 73 in PAO1, 125 in Pf0-1, fold change >2), responsive genes to iron depletion were commonly upregulated in each host. The growth rate of each pCAR1-containing host was lower than that of each pCAR1-free strain in low Fe3+ condition. On the other hand, the mexEFoprN operon encoding an efflux pump of the RND family was upregulated in KT2440 whereas homologous genes in the others were not. We are now analyzing growth phase-dependent alteration in the transcriptome, and it was revealed that the effect of pCAR1 carriage was greater in stationary phase than in log phase in KT2440. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). Y.T. was supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists.


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Two identical copies of a monooxygenase gene in Sphingomonas sp. NP5, which allowed a Pseudomonas putida host strain to degrade nonylphenol isomersMasahiro Takeo1, Yoshihiro Maeda2, Junko Maeda2, Naoki Nishiyama2, Aika Kawasaki2, Chitoshi Kitamura2, Dai-ichiro Kato2 and Seiji Negoro2

1Graduate School of Engineering, Univ. of Hyogo, 2Graduate School of Engineering, Univ. of Hyogo, Japan

Endocrine disrupting chemicals mimic the functions of natural hormones by interacting with their receptors and mainly cause decrease in male fertility and feminization of higher organisms, particularly, fish and aquatic animals. One of such compounds, nonylphenol (NP) has widely contaminated aquatic environments as the degradation product of a non-ionic detergent NP polyethoxylate (1). To understand the biodegradation mechanism, we isolated an NP-degrading bacterium, Sphingomonas sp. NP5 from activated sludge and successfully cloned two identical copies of an NP monooxygenase gene (nmoA) as 8.7-kb and 9.3-kb DNA fragments from this strain. The gene product showed 84 % amino acid sequence identity with the octylphenol 4-monooxygenase of Sphingomonas sp. PWE1 (2). A 1.6-kb DNA fragment encoding nmoA was amplified by PCR and subcloned into a broad host range vector pBBR1MCS-2, and then the resultant plasmid was introduced into Pseudomonas putida KT2440. The cell suspension of the recombinant strain was able to degrade two NP isomers, 4-(1-ethyl-1-methyhexyl)phenol and 4-(1-ethylheptyl)phenol, which were chemically synthesized. HPLC and GC/MS analyses revealed that the former was converted into hydroquinone and 3-methy-3-octanol stoichiometrically, while the latter was converted into nonylhydroquinone and then into nonylbenzoquinone spontaneously, which also decreased gradually. In addition, the cell suspension also oxidized a wide range of alkylphenols, bisphenols, and hydroxybiphenyls, indicating that the NP monooxygenase has surprising broad substrate specificity for these phenolic compounds. Interestingly, p-t-butylphenol, which is suspected of having endocrine disrupting effect, was also converted into major amounts of hydroquinone and t-butylhydroquinone. This suggests that in this degradation, both the release of the side chain from the parent compound and the shift of the side chain to the adjacent carbon atom occurred in parallel.(1) Soares, A. et al. : Environ. Int., 34, 1033-1049 (2008)(2) Porter, AW & Hay, AG : Appl. Environ. Microbiol., 73, 7373-7379 (2007)


Structural basis of electron transfer in aromatic ring-hydroxylating Rieske nonheme iron oxygenases.Takashi Umeda1, Junichi KatsukiI1, Kengo Inoue1, Yuji Ashikawa2, Yusuke Usami1, Haruko Noguchi1, Zui Fujimoto3, Tohru Terada1, Hisakazu Yamane1 and Hideaki Nojiri1

1The University of Tokyo, Tokyo, Japan, 2RIKEN, Hyogo, Japan, 3National Institute of Agrobiological Science, Ibaraki, Japan

Carbazole 1,9a-dioxygenase (CARDO) catalyzes a region- and stereoselective dihydroxylation of various aromatic compounds. CARDO consists of three components: terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase (Red). CARDO is a member of the Rieske nonheme iron oxygenases (RO) that have been grouped into five classes, IA, IB, IIA, IIB and III, according to their number of constituents and the nature of redox centres for electron transfer (ET). We have investigated the molecular mechanism of class-specific ET using four CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Nocardioides aromaticivorans IC177, and Novosphingobium sp. KA1, which are classified into class III, III, IIB and IIA, respectively. Previous investigation on the interchangeability of ET components indicated that ET from Fd to Oxy did not occurred between different classes, while ET from Red to Fd occurred between different class constituents except for class IIA Fd. These results suggest the common feature that Oxy strictly recognize its own Fd as an electron donor. In addition, class IIA Fd specifically recognize its own Red. To investigate the molecular mechanisms of class-specific ET in CARDOs, in this study, we first determined the crystal structures of Oxy, Fd, and Red of class IIA CARDO, and simulated their ET complexes. Then, we compared the simulated structures with previously-solved crystal structure of class III Oxy:Fd complex of CARDO (Ashikawa et al., Structure, 2006) and class IIB Red:Fd complex of biphenyl dioxygenase from Acidovorax sp. KKS102 (Senda et al., JMB, 2007). In both Oxy:Fd complexes, there are surface shape complementation with paired charge distributions in the interacting regions, suggesting that the shape and the charge distribution of the each component are important to form specific Oxy:Fd ET complex. On the other hand, to form the ET complex between Red and Fd, it was concluded that surface shape complementation was only a key determinant, and that surface charge is less important. Accordingly, similar Fd molecule can bind to the Fd-binding regions of Reds in different classes. However, the molecular shape of class IIA Fd is specifically bulky. Docking simulation clearly suggested that this bulky Fd can not bind to other class of Red molecules. This is the reason of the class IIA CARDO-specific strict recognition between Fd and Oxy.


Proteome analysis and phenotypical characterization of an oxyR mutant of the opportunistic pathogen Pseudomonas aeruginosa PAO1Tiffany Vinckx1, Sandra Matthijs1 and Pierre Cornelis1

1Vrije Universiteit Brussel, Brussels, Belgium

Pseudomonas aeruginosa PAO1 is an opportunistic pathogen infecting immunocompromised individuals, especially cystic fibrosis patients. This facultative anaerobe prefers to respire aerobically and hence is challenged with endogenous, oxidative stress due to uncontrolled leakage of electrons from the respiratory chain to oxygen. In P. aeruginosa, oxidative stress is detected and the response mainly coordinated by the LysR-regulator OxyR, stimulating the expression of antioxidant genes katB, ahpC and ahpF [1].In addition to oxidative stress, the presence of oxygen also limits iron availability and iron, in turn, results in the production of dangerous hydroxyl radicals through the Fenton reaction [2]. Under iron-limiting conditions, P. aeruginosa produces 2 known siderophores: pyoverdine and pyochelin [3].Previously, we showed that OxyR inactivation, results in deficient pyoverdine utilization, the primary siderophore of P. aeruginosa. We confirmed that pyoverdine is still taken up by the oxyR mutant (Pa::oxyR), suggesting that the defect is located in downstream processes [4]. In a second part, we studied the influence of OxyR inactivation on the phenotype of this pathogen. We found a two-fold increase of the phenazine pyocyanin, decreased swarming and decreased rhamnolipid production. Although these are all quorum sensing regulated traits and although Pa::oxyR only grows when cell density is high enough, we found no significant change in the production of signal

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molecules, including N-acyl homoserine lactones and the Pseudomonas quinolone signal. In attempts to complement these phenotypes, interesting differences were observed when single or multiple copies of oxyR were present in trans.In a third part, we performed proteome analysis in order to elucidate the fundamental cause(s) of the pyoverdine utilization defect and of the phenotype of this mutant. We could observe an upregulation of Fur and Dps proteins in the oxyR mutant.[1] Ochsner et al. (2000). J Bacteriol 182, 4533-4544 [2] Storz & Imlay (1999) Curr Opin Microbiol 2, 188-194[3] Cornelis & Matthijs (2002) J. Environ. Biol. 4, 787-798[4] Vinckx et al. (2008) FEMS 288, 258-265


Antibiotic tolerance and metabolism of Pseudomonas aeruginosa in an artificial sputum mediumAndrea Wesche1, Sabina Thoma1, Annika Steen1, Piotr Bielecki2, Vitor Martins dos Santos2, Michael Hogardt 3 and Max Schobert1

1Technische Universität Braunschweig, Institute of Microbiology, Spielmannstr.7, 38106 Braunschweig, Germany, 2Systems and Synthetic Biology Group, Helmholtz Center for Infection Research, Inhoffenstrasse 7, 3Max von Pettenkofer-Institut, Aussenstelle Großhadern, Marchioninistrasse 17, D-81377 Munich, Germany

Pseudomonas aeruginosa causes persistent lung infections in the CF lung and is highly resistant to many antibiotics. P. aeruginosa forms biofilm-like microcolonies within mucus-plaques and generates a local microaerobic to anaerobic environment. Biofilm growth and oxygen limitation both contribute to high antibiotic tolerance of this pathogen. To investigate how growth conditions increase antibiotic tolerance under in vitro conditions we used a biofilm-model that simulates respiratory conditions in the CF lung. An artificial sputum medium (ASM) was used to mimic the CF lung habitat and to promote typical microcolony formation of P. aeruginosa. We found that physiological nitrate concentrations of about 250 µM increase the antibiotic tolerance in ASM to different classes of antibiotics, like fluoroquinolones, aminoglycosides, ansamycine and beta-lactame antibiotics. Some of the tested antibiotics as tobramycine and ceftazidime are used for treatment of CF patients with P. aeruginosa airway infection. A comprehensive transcriptome, metabolome and proteome analysis was carried out to characterize the physiology of P. aeruginosa under these conditions leading to increased antibiotic tolerance. Moreover, we compared transcriptome profiles of P. aeruginosa CF isolates from sputum samples with the transcriptome of the same strains grown in ASM to evaluate how well ASM mimics the CF sputum.


Inactivation of reactive oxygen species by the Rubredoxin / Rubredoxin Reductase system in Pseudomonas aeruginosaLutz Wiehlmann1, Claus Urbanke1, Gregor Hageluecken2, Thorsten Adams1, Antje Munder1, Sonja Horatzek1, Wolf-Dieter Schubert2 and Burkhard Tümmler1

1Medizinische Hochschule Hannover, 2Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany

Anaerobic bacteria use the Rubredoxin / Rubredoxin Reductase (Rdx/RdxR) system for the inactivation of reactive oxygen species (ROS) or even the reduction of molecular oxygen to water. In Pseudomonas aeruginosa this system is only known to be necessary for growth on alkanes. We identified the Rdx/RdxR system in a highly virulent CF- isolate as being essential for survival and proliferation after phagocytosis by human polymorphonuclear leucocytes. Additionally, the rdxR mutant exhibited defects in quorum sensing and lower virulence in in vivo infection models (mice and C. elegans). During stationary growth and in in vitro tests, the bacterial cells had a substantially reduced resistance to ROS and had to face a higher oxidative stress (shown by higher levels of cis-methylene fatty acids and increased Fe(III)/Fe(II) ratio). These observed changes could be reverted by complementation in trans and also in in vitro studies by adding the heterologously expressed Rdx and RdxR to a RdxR knock out mutant cell lysate. Transcriptome analyses showed that the Rdx/RdxR system is upregulated in several highly virulent CF isolates under high oxidative stress and in the stationary growth phase. Biochemical analyses and X-ray crystallography showed that the Rdx/RdxR system transfers electrons with very high efficiency to oxidized iron centres, keeping them in the reduced, reactive state. The reaction of reduced RdxR with its reaction partners is almost diffusion controlled, enabling P. aeruginosa not only to keep proteins with iron centres in an active state during oxygen stress but also to gain energy from the degradation of ROS.These findings support the hypothesis that the Rdx/RdxR system is a highly efficient P. aeruginosa defence mechanism which minimizes oxidative stress and thus facilitates persistence and proliferation in infected hosts.


Isolation and identification of the lysR gene in Comamonas testosteroniGuangming Xiong1, Wenjie Gong1 and Edmund Maser1

1Institute of Toxicology and Pharmacology for Natural Scientists, Kiel, Germany

Comamonas testosteroni 3a-hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) is a key enzyme in the degradation of steroid compounds and polycyclic aromatic hydrocarbons in soil, and may therefore play a significant role in the bioremediation of harmful compounds in the environment. We previously reported the cloning and isolation of two repressors and an activator which regulate the gene expression of 3a-HSD/CR in C. testosteroni. After steroid induction 3a-HSD/CR gene expression increased. In the present study, we have identified the lysR gene as another and new activator gene for 3a-HSD/CR gene regulation. LysR was isolated from the chromosomal DNA of C. testosteroni. The lysR gene consists of 912 bp and locates 3.360 kb downstream from the 3a-HSD/CR gene. To produce purified LysR protein, the lysR gene was cloned into plasmid pET-15b and the overexpressed protein purified by its His-tag sequence on metal chelate chromatography. SDS PAGE revealed a molecular mass of the LysR protein of 33.6 kDa (303 aa). For functional studies and co-transformation experiments, the Tac promoter was cloned upstream of the lysR gene into pK18 to yield plasmid pKtac-lys1. Interestingly, 3a-HSD/CR expression increased when pKtac-lys1 was cotransformed with plasmid pAX1 (harbouring the 3a-HSD/CR gene) into E. coli HB101. We then produced knock-out mutants of the lysR gene in C. testosteroni. As expected, these knock-out mutants expressed low levels of 3a-HSD/CR, even after testosterone induction. From these results we conclude that lysR is an important player in the regulation of the steroid degradation pathway in C. testosteroni.

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Functional analyses of H-NS-like proteins in Pseudomonas putida KT2440 harbouring IncP-7 plasmid pCAR1Choong-Soo Yun1, Kunihiko Naito1, Toshiharu Takeda1, Masaki Shintani1, Hiromi Nishida2, Hisakazu Yamane1 and Hideaki Nojiri1

1Biotechnology Research Center, The University of Tokyo, 2Professional Program for Agricultural Bioinformatics, The University of Tokyo, Japan

H-NS is one of the major nucleoid structuring proteins associated with the bacterial chromosome, known as a global regulator which influences the expression of many genes in a lot of Gram-negative bacteria. Moreover, it has been also known that some bacteria have an additional H-NS protein encoded on the plasmid. And this additional member of the H-NS protein family also influences the expression of many genes existing on both chromosome and plasmid. Pseudomonas putida KT2440 has five H-NS-like genes on the chromosome. And IncP-7 plasmid pCAR1 has an H-NS-like gene named pmr (plasmid encoded MvaT-like regulator). pCAR1 is a 200-kb self-transmissible plasmid involved in carbazole degradation. When pCAR1 is conjugally transferred into KT2440, an additional sixth member of H-NS-like regulator, Pmr, may provide very unique circumstance and modulate the global regulation by the chromosomally-encoded H-NS-like regulators. To clarify the cross-communication via both chromosome- and plasmid-borne H-NS like regulators, we constructed the mutants of KT2440 or KT2440 (pCAR1), in which each H-NS-like regulator gene was replaced with a C-terminal 6 x His-tagged form. Using these strains, we investigated the expression levels of H-NS-like regulators during the growth on succinate by Western blot analyses. As the results, among the six members, Pmr, PP-1366 (TurA) and PP-3765 (TurB) were detected as major components through the growth under our experimental conditions.On the other hand, complete genome sequences of KT2440 chromosome and pCAR1 enable us to design a customized high-density oligonucleotide tiling array. We constructed the deletion mutants for six H-NS-like regulator genes, and the mRNA expressed in each mutant was mapped to the high-density tiling array chip (RNA mapping). Transcriptome comparison using RNA mapping data of KT2440, KT2440 (pCAR1), and KT2440 pmr) revealed that 55 % of the genes whose transcription levels∆ pCAR1 were affected by the carriage of pCAR1 (fold change > 2) was through Pmr function, although the regulatory mechanism by Pmr should be clarified. Transcriptome analyses of chromosomally H-NS-like regulator gene-knockout mutants of KT2440 and KT2440 (pCAR1) are currently underway. In addition, the results of genome-wide survey of the binding sites of H-NS-like regulators by chromatin affinity purification coupled with high density tiling chip (ChAP-chip) analyses will be reported.


Metabolome analysis of Pseudomonas aeruginosa using gaschromathography-mass spectrometry Eliane Frimmersdorf1, Sonja Horatzek2, Lutz Wiehlmann2, Kerstin Schreiber1, Dietmar Schomburg1

1Institute of Bioinformatics & Biochemistry, Spielmannstr. 7, 38106 TU Braunschweig, 2Medical School of Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Metabolome analysis is a highly sensitive method for phenotype characterization. It indicates changes in the cellular state caused by genetic or environmental perturbations. The comprehensive knowledge on intracellular metabolite concentrations can be used for a detailed comparison for the assessment of virulence of pathogens or for strain improvement in industrial processes. Here we present the results of a metabolome analysis of Pseudomonas aeruginosa. The standard procedure for bacterial metabolome analysis using gaschromatography coupled to mass spectrometry developed in our lab was applied (Strelkov et al. 2004; Börner et al. 2007). Different cystic fibrosis isolates, transposon mutants with lowered quorum sensing activities and two labratory strains were analysed. The comparison of the metabolic profiles can be used to identify metabolites that are possibly involved in the pathogenicity of the strains. Two different media were applied for the cystic fibrosis (CF) isolates; a minimal medium supplemented with glucose and methionine, and a minimal medium supplemented with amino acids to simulate the lung environment in which many amino acids are present. The growth behaviour in the two media differed slightly between the CF isolates and the labratory strains. Metabolome analysis showed increased concentrations of some amino acids in the CF isolates.For the transposon mutants with lowered quorum sensing different C-sources (glucose, succinate, citrate, cadaverine) were tested in minimal medium. During growth on glucose and succinate the transposon mutants and the lab strains accumulated the polyamine pathway metabolites (e.g. 5-methylthioadenosine). Interestingly, in the presence of glucose the transposon mutants show enhanced concentrations of metabolites involved in the fatty acid metabolism (e.g. 2-monooleoylglycerol, palmitic acid amide). When growing on citrate the transposon mutants and the lab strains increased pools of the glyoxylate shunt metabolites were found.

Translational Research

Oral presentations

O-05 Topic: Translational Research

New insights into the Pseudomonas putida KT2440 reactomeManuel Ferrer1, Maria E. Guazzaroni1, Jose M. Vieites1, Florencio Pazos2 and Ana Beloqui1

1CSIC - Institute of Catalysis, 2CSIC - Centro Nacional de Biotecnología, Madrid, Spain

It is not generally accepted that conceptual advances in microbial science will not only rely on the availability of innovative sequencing platforms but also on sequence-independent tools for getting an insight into the functioning of pure organisms and microbial communities. The reasons are clear. First, every single cell or environmental genomic project added a huge number of putative genes, the function of which is often unknown and at best deduced from sequence comparison. Second, even the best annotations only created hypotheses of the functionality and substrate spectra of proteins which require experimental testing by classical disciplines such as physiology and biochemistry. This highlighted the difficulties of making sense of sequence data: a significant proportion of the open reading frames could not be characterized because there were no similar sequences in the databases. We described here a new metabolite

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array for genome sequence-independent functional analysis of metabolic networks, the reactomes, of organisms and communities. Its potential use in Systems Biology is described.

O-06 Topic: Translational Research

Chemical biology, biofilms and PMN-Leukocytes Michael Givskov1

1University of Copenhagen, Denmark

The biofilm lifestyle plays a key role in many chronic bacterial infections. Pseudomonas aeruginosa is known as a notorious biofilm former. During establishment, bacteria emerge in a “harmless” state (no expression of virulence factors) as they increase in numbers and build biofilms. In the biofilm mode, P. aeruginosa uses “quorum sensing or QS” communication to inform its fellow bacteria about progress in the infectious process. When the QS system signals, the biofilm launches a cocktail of destructive virulence factors which in turn attract phagocytic white blood cells (PMNs), a major cellular component of the innate immune system. To offer protection, a rhamnolipid based shield is also launched which on contact kills attaching PMNs. Work in progress indicates that cross communication between bacteria and immune system are important in these Eukaryote–Prokaryote interactions. Small molecule signal blockers (QS inhibitors) can be introduced which do not kill bacteria but block communication. The shield is not formed and the PMNs can actively break down the bacterial biofilm. QS inhibitory chemistry is available in a variety of ecological niches from coral reefs to herbs.


PO-73 Topic: Translational Research

Inhalation of acid sphingomyelinase inhibitors normalizes ceramide, inflammation and Pseudomonas aeruginosa infections in cystic fibrosis Katrin Anne Flegler1, Anja Lüth2, Burkhard Kleuser2 and Erich Gulbins1

1Dept. of Molecular Biology, University of Duisburg-Essen, Essen, 2Dept. of Pharmacy, Pharmacology and Toxicology, Free University Berlin, Berlin, Germany

We have recently demonstrated a critically involvement of sphingolipids in cystic fibrosis. CFTR-knock-out or B6.129P2(CF/3)-CFTRTgH(neoim)Hgu mice (named CFMHH) that produce low levels of CFTR accumulate ceramide in their lungs. Ceramide accumulation seemed to be mediated by an imbalance between the activities of the acid sphingomyelinase and acid ceramidase in a subpopulation of cellular vesicles that are more alkaline in CFTR-deficient than in wild type cells. Increased concentrations of ceramide triggered enhanced cell death of respiratory epithelial cells, release of pro-inflammatory cytokines in the lung, release of DNA into the mucus, chronic inflammation and high sensitivity to P. aeruginosa infections. The findings were confirmed in human post-mortem specimen that also revealed high levels of ceramide in lung tissue of cystic fibrosis patients. Here, we analysed by mass spectrometry, fluorescence microscopy and biochemical measurements ceramide levels in lung epithelial cells and macrophages of cystic fibrosis mice. All three methods demonstrate a significant increase of total pulmonary ceramide in CFMHH mice. Ceramide accumulated in both, bronchial epithelial cells and macrophages. Further biochemical and fluorescence microscopy studies also reveal a marked accumulation of ceramide in the small intestine, an organ, which is also severely affected in cystic fibrosis. Next, we determined whether we are able to normalize pulmonary ceramide levels, inflammation and infection susceptibility in CFMHH mice by interfering with the pulmonary sphingolipid metabolism. We demonstrate that inhalation of five different inhibitors of the acid sphingomyelinase reduces ceramide levels in bronchial epithelial cells of CFMHH mice. Most important, inhaled acid sphingomyelinase inhibitors reduced inflammation in the lung of CF-mice and prevented infection with P. aeruginosa. In addition, inhibition of the acid sphingomyelinase by an un-physiological increase of cholesterol upon feeding the mice with Peptamen diet also reduced pulmonary ceramide levels. To gain some insight into the molecular mechanisms of ceramide-mediated alterations in CF, we investigated molecular mechanisms of the induction of apoptosis by Pseudomonas aeruginosa in wild type and CF-cells.In summary, the studies indicate that inhalation of drugs that inhibit the acid sphingomyelinase and normalize pulmonary might be beneficial for cystic fibrosis with minimal side effects.

PO-74 Topic: Translational Research

Genomic mining for bioactive small molecules in the genome of Pseudomonas fluorescens Pf-5 and Pseudomonas syringae pv. syringae B728aHarald Gross1

1University of Bonn, Germany

Through genome sequence analysis, it had become evident that Pseudomonads contain a much higher genetic capacity to produce secondary metabolites than expected, and that many of the products of these genetic pathways remain unknown at the present time. Since numerous microbial secondary metabolites display important biological activities, the exploration and utilization of these compounds is promising and might lead to unique natural products with pharmaceutical potential.Both, P. fluorescens Pf-5 and P. syringae pv. syringae B728a (Pss B728a) got sequenced in 2005 due to their significance in the agricultural area: Pf-5 is known as a biological control agent because it suppresses crop diseases caused by soilborne plant pathogens whereas Pss B728a is known as a plant pathogen that causes brown spot disease on snap bean plants. Bioinformatic analyses of the sequenced genome revealed a large number of orphan pathways encoding secondary metabolites in addition to known biosynthesis clusters. Using different genomic guided approaches, we were able to characterize some of these pathways with natural products, e.g. the new antifungal natural products orfamides A-C and new cytotoxic rhizoxin-derivatives from Pf-5. Furthermore, the genetic locus in Pss B728a for the production of the compatible solute N-acetyl-glutaminyl-glutamine-amide could be annotated.

PO-75

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Topic: Translational Research

Green production of substituted aromatics by engineered solvent-tolerant Pseudomonas putida S12Harald J Ruijssenaars1 and Han De Winde2

1TNO Quality of Life, 2 Delft University of Technology, Faculty of Applied Sciences, Dept of Biotechnology, The Netherlands

Pseudomonas putida S12 thrives in minimal medium with a second phase of toluene or 1-octanol. Its exceptional solvent tolerance makes this strain pre-eminently suited for the production of industrially relevant, but toxic chemicals such as substituted aromatics. We have engineered P. putida S12 into a versatile whole-cell biocatalyst with regard to substrate utilization and product formation. An array of substituted aromatics such as phenol, p-hydroxystyrene, cinnamate and p-hydroxybenzoate can be produced from renewable feedstock such as glucose and (raw) glycerol (1-4). The products are synthesized via aromatic amino acids (L-phe and L-tyr), utilizing combined heterologous and endogenous (partial) pathways. The initially modest production characteristics were improved by orders of magnitude by random mutagenesis and high-throughput screening (1, 2). Transcriptomics, proteomics and metabolic flux analyses on improved mutants demonstrated that the aromatic amino acid biosynthesis level was considerably enhanced and furthermore provided useful leads for additional targeted optimization of the production hosts (2). In addition to optimizing productivity and expanding product range, the efficient utilization of biomass-derived feedstock is investigated. Wild-type P. putida S12 readily metabolizes the model renewables glucose and glycerol, but real-life lignocellulose hydrolysate also contains considerable amounts of pentoses and other substances that P. putida S12 cannot utilize. P. putida S12 strains were constructed that efficiently utilize pentoses either via a phosphorylative (5) or an oxidative pathway. In addition, P. putida S12 strains have been constructed that metabolize other compounds present in lignocellulose hydrolyzates. The substrate utilization and aromatics production properties are currently combined into single strains for efficient conversion of cheap biomass-derived feedstock to value-added aromatic products.

1. Nijkamp, K., Van Luijk, N., De Bont, J.A.M., and Wery, J. Appl. Microbiol. Biotechnol. 69, 170-177. 2005.2. Wierckx, N.J.P., Ballerstedt, H., De Bont, J.A.M., De Winde, J.H., Ruijssenaars, H.J., and Wery, J. J. Bacteriol. 190, 2822-30. 20083. Verhoef, S., Ruijssenaars, H.J., De Bont, J.A.M., Wery, J. J. Biotechnol. 132, 49-56. 2007.4. Verhoef, S., Wierckx, N.J.P., Westerhof, R.G.M., De Winde, J.H., Ruijssenaars, H.J. Appl. Environ. Microbiol. 75, 931-9. 2009.5. Meijnen, J.-P., De Winde, J.H., Ruijssenaars, H.J., Appl. Environ. Microbiol. 74, 5031-7. 2008.

P-76 Topic: Translational Research

Highly motile Pseudomonas fluorescens F113 mutants with a reduced ability to form biofilms on abiotic surfaces show better biocontrol performance in the Phytophthora cactorum-strawberry system.Emma Barahona1, Ana Navazo1, Miguel Redondo-Nieto1, Francisco Martinez-Granero1, Marta Martin1 and Rafael Rivilla1

1Universidad Autonoma de Madrid, Spain

Motility is one of the most important traits in rhizosphere colonization by P. fluorescens F113, being mutants with reduced motility very poor competitors. We have also shown that rhizosphere selects hypermotile phenotypic variants, which displace the wild type strain in competitive analysis. It has been described that the aggregation of bacteria on the root surface is a consequence of biofilm formation that it is supported by a matrix produced by the bacteria. Recent reports oppose a planktonic lifestyle characterized by motility to a sessile lifestyle in the form of biofilm. The transition between both lifestyles is regulated by the levels of cyclic di-guanilate (di-GMPc), and these levels are controlled by diguanilate cyclases (proteins with a GGEDF domain) and phosphodiesterases (proteins with EAL or HD domains). In these sense, mutants unable to form biofilms show reduced levels of di-GMPc and increased motility. To test the hypothesis of biofilm formation during rhizosphere colonization, we have analyzed the abilities to form biofilms of a hypermotile variant selected in the rhizosphere and several hypermotile mutants. All of them show impairment in biofilm formation, however, they displace the wild type strain in competitive colonization analysis. Therefore, in P. fluorescens F113 biofilm formation is not necessary for rhizosphere colonization. Furthermore, biocontrol tests done in Phytophthora cactorum/strawberry system have shown that the most hypermotile mutant is the best strain in blocking the symptoms produced by the pathogen in the plant.


Development of a mucosal vaccine against Pseudomonas aeruginosaUlrich Baumann1, Dirk Bumann2 and Bernd-Ulrich von Specht3

1Hanover Medical School, 2Biozentrum, University of Basel, Switzerland, 3Centre for Clincal Research, University Hospital, Freiburg, Germany

Background: Pseudomonas aeruginosa is a leading pathogen for morbitiy and mortality in patients with airway disease, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), or artificial ventilation. Despite major advances in antibiotic therapy preventive measures remain desirable.Objective: To develop an antipseudomonal vaccine with particular emphasis on airway protection.Methods: experimental and clinical studies with newly developed oral, nasal and systemic vaccines based on a recombinant fusion protein of the outer membrane proteins OprF and OprI.Results: A nasal gel OprF-OprI vaccine and an attenuated oral Salmonella typhimurium live vaccine expressing OprF-OprI engendered considerably higher levels of antibodies recovered at the respiratory mucosa in nasal wash and brochoalveolar lavage (BAL) than a conventional systemic OprF-OprI almun vaccine in mice. To facilitate detection of antibodies in the lower airways, we established the induced sputum technique as a non-invasive measure. In 3 clinical studies the nasal OprF-OprI vaccine and 4 newly constructed attenuated live Salmonella live vaccines based on strains Ty21a and CVD induced a prounced airway antibody response.Conclusion: Mucosal vaccination is a promising strategy for airway protection against P. aeruginosa.Supported by DFG, ZIT, Mukoviszidose e.V., and Appenroth-Stiftung

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Antibacterial efficacy of phages towards Pseudomonas aeruginosa infections in mice and Drosophila melganogasterHyun-Hee Jung1, Yun-Jeong Heo1, Yu-Rim Lee1, JungEun Kim2, GwangPyo Ko2 and You-Hee Cho1

1Sogang University, 2 Seoul National University, South Korea

Phage therapy against Pseudomonas aeruginosa infections has received renewed attention owing to the increasing prevalence of antibiotic resistance in this bacterium. Here, we isolated and characterized two new potentially lytic bacteriophages (MPK1 and MPK6), which produced large and clear plaques on P. aeruginosa strain PAO1. Based on their morphology, MPK1 belongs to the Myoviridae, while MPK6 belongs to the Podoviridae. The B-polysaccharide of lipopolysaccharide was required for infection, suggesting their hostspectra are associated with the serotypes of the P. aeruginosa strains. Intramuscular and intraperitoneal administration of MPK1 and, to the lesser extent, MPK6 significantly protected mice from mortality caused by PAO1-induced peritonitis-sepsis (p < 0.01). Mice treated with one of both phages also had the lower bacterial burdens in liver, lung, and spleen. The antibacterial efficacy of MPK1 and MPK6 was also evaluated based on Drosophila melanogaster systemic infection caused by P. aeruginosa, for which phages were administered by feeding. Both phages significantly delayed the PAO1-induced killing of D. melanogaster (p < 0.001), although MPK1 persisted longer than MPK6 in uninfected D. melanogaster tissues. These results suggest that mini-scale experiment using D. melanogaster infection is valid to evaluate the antibacterial efficacy for phage therapy towards P. aeruginosa infections.


Heterogeneity of heat resistant proteases from milk spoiling Pseudomonas spp.Sophie Marchand1, Gonzalez Vandriesche2, An Coorevits3, Katleen Coudijzer4, Valerie De Jonghe4, Koen Dewettinck5, Paul De Vos3, Bart Devreese2, Marc Heyndrickx4 and Jan De Block4

1Institute for Agricultural and Fisheries Research (ILVO) , 2Ghent University, Faculty of Science, Department of Biochemistry, Physiology and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, 3Ghent University, Faculty of Science, Department of Biochemistry, Physiology and Microbiology, Laboratory of Microbiology, 4Institute for Agricultural and Fisheries Research (ILVO T&V), 5Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Belgium

Proteolytic psychrotolerant bacteria, predominantly Pseudomonas spp. still cause significant losses for the dairy industry, due to the production of thermostable proteases in raw milk. A polyphasic identification approach led to the identification of P. fragi and P. lundensis as predominant milk spoilers (53% of 103 proteolytic isolates) in 89 Belgian raw milk samples. In general, the aprX gene, encoding an alkaline metalloprotease, is believed to be the key feature in proteolytic spoilage by pseudomonads. However, up to now the presence of the aprX gene was not yet demonstrated in P. fragi and P. lundensis. To evaluate the presence of the aprX gene within Pseudomonas members, 29 Pseudomonas milk strains and 26 Pseudomonas reference strains were screened for the presence of the aprX gene in their genome. Although zymogram analysis revealed that all Pseudomonas milk strains produced heat stable proteases with a similar molecular weight, analysis of the protease sequences on the DNA and the protein level revealed a high heterogeneity within these heat stable Pseudomonas proteases. The obtained data revealed some interesting features for the development of identification and detection methodologies for pseudomonads in milk. Moreover, our data revealed that interspecies variations within the aprX gene could contribute to a molecular classification framework for Pseudomonas spp., in particular within the heterogeneous Pseudomonas fluorescens group.


The role of lymphocytes in response to a Pseudomonas aeruginosa vaccineJennifer M. Scarff1, Antonio DiGiandomenico1 and Joanna Goldberg1

1University of Virginia, Charlottesville, USA

Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the Pseudomonas aeruginosa lipopolysaccharide O antigen was effective in preventing P. aeruginosa colonization and mortality in wild-type mice after intranasal challenge with a lethal dose of bacteria. We also showed that the hypersusceptibility seen in leukopenic and neutropenic mice was reduced when mice were vaccinated or received immune serum at the time of infection, suggesting an important role for neutrophils and antibodies in response to challenge. However, the cells required for the generation of the protective immune response after vaccination remain unknown. After vaccination, mice lacking mature B (muMT) or T (nu/nu) cells produced either no antibodies or low amounts of Pseudomonas-specific IgM, respectively. Neither response was capable of protecting these mice from a P. aeruginosa challenge. However, both the muMT and nu/nu mice were protected from infection when immune serum was administered intranasally at the time of infection, indicating that the defect is in the ability of these mice to generate antibody, not in responding when antibody is already present. To further investigate the role of T cell subsets in the development of a protective antibody response, CD4-/- and CD8-/-

mice were vaccinated. Interestingly, both the CD4-/- and CD8-/- mice produced a robust, specific IgG response after vaccination and were protected from subsequent challenge. In support of these findings, vaccinated BALB/c mice whose CD4 cells were removed by monoclonal antibody GK1.5 also survived lethal P. aeruginosa challenge. Altogether, these data suggest that while both B and T cells are required for the formation of a protective vaccine-induced antibody response, the lack of either CD4 or CD8 cells does not abrogate the formation of this immune response. Also, for this vaccine strategy, CD4 cells are dispensable after an immune response has been generated. Further studies characterizing the role of CD4 and CD8 cells by transferring naive cells into athymic mice prior to vaccination are ongoing. We are also transferring cells from vaccinated mice into naive mice and looking for protection to transfer. These data indicate the importance of T cells in the formation of a robust antibody response to the O antigen of our vaccine, an antigen typically associated with a T cell-independent antibody response.

Systems and Synthetic Biology

Oral presentations

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O-07 Topic: Systems and Synthetic Biology

Metabolic comparison of the in-silico phenotype-genotype relationship of Pseudomonas putida and Pseudomonas aeruginosaVitor A.P. Martins dos Santos1, Jacek Puchalka2, Matthew Oberhardt3, Manuel Ferrer4, Miguel Godinho2, Piotr Bielecki2 and Jason Papin3

1Helmholtz Center for Infection Research, 2Helmholtz Center for Infection Research, 3Department of Biomedical Engineering, University of Virginia, Charlottesville, USA, 4Institute of Biocatalysis, CSIC, Madrid, Spain

The relationship between the genotype and the phenotype is complex, highly non-linear and cannot be predicted from simply cataloguing and assigning gene functions to genes found in a genome. Comprehensive understanding of cellular metabolism requires placing the function of every gene in the context of its role in attaining the set goals of a cellular function. This demands the integrated consideration of many interacting components. Mathematical modelling provides us a powerful way of handling such information and allows us to effectively develop appropriate frameworks that account for these complexities.We report here an in silico representation of Pseudomonas putida (a versatile soil bacterium of biotechnological importance) and Pseudomonas aeruginosa (a threatening pathogen) that describes their metabolic capacities within the scope of their environmental constraints. Using annotated genome sequence data, biochemical information and strain-specific knowledge, we analysed, interpreted (and we expect to ultimately predict) the cellular behaviour of these micro-organisms under a range of conditions relevant for both human health and environmental applications. This was done using flux-balance-analysis related methods for the entire metabolic networks of these bacteria. The networks comprise between 700 and 900 reactions representing between 600 and 850 genes, which is about 12 to 14% of the genome of these bacteria. Preliminary results suggest that the metabolism of P. aeruginosa may be more flexible than that of P. putida for the considered data set. This is an emergent property of the system that could not be predicted solely on basis of the linear comparison of gene lists. The construction of comprehensive metabolic maps provides a framework to study the consequences of alterations in the genotype and to gain insight into the phenotype-genotype relation. Ultimately, this analysis defines the entire metabolic space of the possible flux distributions and metabolic interactions within the network. A direct comparison of this phenotypic space for both bacteria will possibly help in identifying orphan genes, evolutionary features and genetic plasticity. The use of such models to choose the most informative knockouts and ultimately, to rationally design experiments relevant for the elucidation of the behavior of these bacteria in polluted environments or within the scope of their relationships with an infected host will be discussed.

O-08 Topic: Systems and Synthetic Biology

Systems modeling of Pseudomonadales: biotechnological and biomedical applications.Ines Thiele1

1Center for Systems Biology, University of Iceland

Systems Biology is a rapidly growing discipline. It is widely believed to have a broad transformative potential on both basic and applied studies in the life sciences. In particular, metabolic network reconstructions play a key role as they provide a framework for investigation of the mechanisms underlying the genotype-phenotype relationship. The metabolic reconstructions of Pseudomonas putida and Pseudomonas aeruginosa have been recently published. The general process to reconstruct genome-scale metabolic networks is introduced. The content of two metabolic networks is compared and possible biotechnological and biomedical applications are discussed.


PO-81 Topic: Systems and Synthetic Biology

SCaRAB – Systems Biology of carbon redirection in alginate biosynthesisSven Even F Borgos1, Per Bruheim2, Elon Correa3, Annette Dreisbach4, Trond E Ellingsen5, Helga Ertesvåg2, Manuela Gesell-Salazar4, Royston Goodacre3, Elke Hammer4, Øyvind M Jakobsen5, Geir Klinkenberg5, Stina K Lien2, Jens Nielsen6, Marianne B Nilsen2, Frank Schmidt4, Mona Senneset5, Håvard Sletta5, Yi-Qian Sun2, Uwe Völker4 and Svein Valla2

1Norwegian University of Science and Technology, 2Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway, 3School of Chemistry, University of Manchester, Manchester, United Kingdom, 4Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald , Germany, 5Department of Biotechnology, SINTEF Materials and Chemistry, Trondheim, Norway, 6Systems Biology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

Bacterial alginate is an exopolysaccharide produced by Azotobacter vinelandii and some Pseudomonas spp. In the opportunistic pathogen P. aeruginosa, the alginate-producing (mucoid) phenotype is commonly found in the lungs of cystic fibrosis patients, where it causes severe and often fatal infections with high resistance to antibiotic treatment. Our strain of study, P. fluorescens SBW25, harbours the alginate biosynthetic genes but does not produce the polysaccharide under any of the conditions investigated. Activation of alginate biosynthesis has been obtained by introducing, into the regulatory gene mucA, the same mutation that is very commonly found in mucoid P. aeruginosa. This induces a carbon drain towards alginate, originating from fructose-6-phosphate, that can approach 40 % of the cellular carbon uptake. Mutants with continuously graded induction of alginate biosynthesis have also been constructed based on the Pm promoter from P. putida.

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We are coordinating a European Systems Biology project on using alginate biosynthesis in P. fluorescens SBW25 as a model system for studying metabolic network control and robustness, including the use of this strain as a non-pathogenic model system for P. aeruginosa. The wet-lab part of this includes chemostat cultivations on several carbon sources with analysis of the full -ome complement; transcriptome, proteome and metabolome including fluxome. To aid the analysis, a transposon mutant library of 11000 mutants has been constructed and screened for altered phenotype. Integration of data is done on the scaffold of a reconstructed genome-scale stoichiometric model based on the genome sequence and annotation of the organism, as well as complementary approaches including genetic algorithms and Bayesian networks. Reporter algorithm analysis will be performed, based on transcriptome data and the genome-scale reconstruction, to identify highly regulated subnetworks and key (reporter) metabolites.Thus far, we have observed profound, and mostly correlated, effects on the transcriptome and proteome from induction of alginate biosynthesis; metabolome studies are under way. We have also been able to start isolating and analysing the pleiotropic effects on the -omes from the MucA regulator knock-out, other than those directly related to alginate.


Synthetic genetic tools for post-genomic analysis and engineering of PseudomonasEsteban Martínez-García1, Miguel Arévalo2 and Victor de Lorenzo1

1Centro Nacional de Biotecnología-CSIC, 2BioMedal SL, Sevilla, Spain

The goal of Systems Biology is to obtain a complete view of the cell. Gram-negative bacteria, in particular the genus Pseudomonas provide optimal experimental organisms for this endeavor, as they present a generally short generation time, reduced genetic complexity and can be examined with a considerable variety of molecular tools. Apart of the classical insertional mutagenesis the functioning of complex regulatory networks is revealed only when perturbations -rather than sheer knockouts- are entered in a dynamic system. Yet, the vector toolbox for this type of system analysis in bacteria other than E. coli has remained basically unchanged for the last 20 years (1). In this context, we have developed two genetic tools that broaden the range of operations that can be effected on Pseudomonas and other Gram-negative bacteria. First, we have designed a new I-SceI-based chromosomal engineering method for Pseudomonas putida that allows multiple deletions or insertions of chromosomal sequences of choice, as well as an unlimited number of allelic replacements in the genome. The key step in the process is the transient expression of the I-SceI endonuclease, that recognizes and cleaves an 18 bp sequence producing a double strand break that cells are forced to repair by homologous recombination (2,3). To this end, the R6K-suicide vector pJP5603-ISceIv2 is used as a frame for adding two I-SceI recognition sites to the DNA segment of interest inserted in its polylinker. The I-SceI enzyme is then provided by the plasmid pSW/I-SceI (4). This vector system has been instrumental for deleting all four prophages found in the chromosome of P. putida KT2440: prophage-1 (PP3849-PP3920), prophage-2 (PP3026-PP3066), prophage-3 (PP2266-PP2297) and prophage-4 (PP1532-PP1586), thereby creating a phage-free P. putida strain that is optimized for engineering purposes. The second tool is the plasmid pBAM1, a synthetic and updated version of the classical pUT/mini-Tn5 Km1 (1). The sequence has assembled in a modular fashion and it has been altogether edited for eliminating every feature of the original design that was detrimental for its utilization. The plasmid can be delivered by mating, giving an average insertion frequency of the corresponding transposon of 1.8*10-3. In addition, pBAM1 can be electroporated as well, with a high efficiency of mutants (1*10-7).References(1) de Lorenzo et al. (1990)(2) Posfai et al. (1999)(3) Flannagan et al. (2008)(4) Wong and Mekalanos (2000)


Integrating genome-scale transcriptional regulation and metabolism in Pseudomonas aeruginosa elucidates metabolic phenotypes relevant to its virulenceJason A. Papin1 and Matthew Oberhardt1


Network analysis has emerged as a powerful tool for characterizing microbial systems. The recent publication of the genome-scale reconstruction for P. aeruginosa has been used to characterize key features of its metabolic capabilities, predict gene essentiality on a genome-scale with nearly 85% accuracy, and probe fundamental biological questions. The transcriptional regulatory network of P. aeruginosa has now been reconstructed and integrated with the existing metabolic model, accounting for the function of approximately 1300 genes. This integrated reconstruction of metabolism and transcriptional regulation is the largest such model of a pathogen to date and it enables a significant improvement in the ability to predict metabolic phenotypes of this pathogen. This integrated model has now been used to interpret the metabolic phenotypes of mutants identified through signature-tagged mutagenesis screens, facilitating the characterization of key metabolic phenotypes relevant to the virulence of the pathogen. This represents a first major step towards comprehensive, systems-level understanding of pathogenicity in this dangerous organism.

P-84

Removed by the author before online publication.

P-85 Topic: Systems and Synthetic Biology

Optimisation of P. putida KT2440 JD1 as a host for the production of cis, cis muconate from benzoateJoost van Duuren1, Vitor Martins dos Santos2, Astrid Mars3, Jan Wery4 and Gerrit Eggink3

1System and Synthetic Biology, Helmholtz Zentrum fur Infektionsforschung (HZI), 2System and Synthetic Biology, Helmholtz Zentrum für Infektionsforschung (HZI), 3Wageningen Universiteit, Agrotechnology & Food Sciences Group, Business Unit Biobased Products, 4Dyadic Nederland BV

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The main objective of this project is to optimize Pseudomonas putida as a whole cell host for the cofactor dependent production of fine chemicals. P. putida is a versatile non pathogenic bacterium. As the genome sequence of P. putida KT2440 is available, it is possible to use global genomics techniques for a better understanding and optimization of the whole cell biocatalyst. In this project, the conversion of benzoate into cis, cis muconate by a mutant of P. putida KT2440 is studied. The metabolite cis, cis muconate contains a dicarboxylic acid structure with conjugated double bounds, which can be used as a commodity for the production of Nylon 6,6 by hydrogenation to adipic acid.The mutant P. putida KT2440 JD1 was obtained by using NTG mutagenesis. It converts benzoate to almost stoichiometric amounts of cis, cis muconate, which accumulates in the culture medium. Transcriptomic analysis revealed that of the two genes (PP3166 and catA) encoding catechol 1,2 dioxygenase on the genome of P. putida KT2440, only gene PP3166 was transcribed in the mutant. The cat operon on which gene catA is situated could not be upregulated anymore by P. putida KT2440 JD1 as a result of a point mutation in the transcriptional regulator gene catR.The toxicities of cis, cis muconate and benzoate have been tested in batch cultures of P. putida KT2440 JD1 in the presence of increasing amounts of benzoate and/or cis, cis muconate. The results show that benzoate is approximately 14 times more toxic than cis, cis muconate and that the combination of both components has a cumulative toxicity effect. Out of these toxicity experiments, it was also determined that P. putida KT2440 JD1 is at least able to survive in 5 mM benzoate and 350 mM cis, cis muconate. In order to keep the benzoate concentration low and to avoid the accumulation of an intermediate metabolite, cis, cis muconate was produced via a pHstat where two hydrogen atoms are formed in the last conversion step from catechol to cis, cis muconate. The pHstat was performed under controlled growth conditions with a growth rate of 0.04, whereby a final concentration of 130 mM cis, cis muconate was reached. Catechol was measured in the medium during the pHstat. It has also been shown that catechol down regulates the cat operon using RT PCR. Therefore P. putida KT2440 JD1 has been optimized by bringing gene catA back into the cell situated on a plasmid under the control of a strong promoter.


A Systems Biology approach for studying the polyhydroxyalkanoate metabolism in Pseudomonas putida KT2442Isabel F. Escapa1, Laura de Eugenio1, Juan Nogales1, Eduardo Diaz1, Jose Luis García1 and Mª Auxiliadora Prieto1

1Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including pseudomonads, which are accumulated in the cytoplasm as carbon and energy storage material when culture conditions are unbalanced. One of the PHAs roles that have been classically considered is to act as sink for carbon and reducing equivalents when other nutrient supplies are limited (1). Bacteria facing carbon excess and nutrient limitation store the extra carbon in form of PHA via PHA polymerase (PhaC). Under carbon starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases 3-hydroxycarboxylic acid monomers. Recently, it has been proposed in Pseudomonas putida GPo1 that the released monomers are then activated to hydroxyacyl-CoAs by an acyl-CoA-synthetase (ACS1) associated with the PHA granules in an ATP-dependent reaction (2). Hydroxyacyl-CoAs are substrates for PHA polymerase as well as intermediates for the fatty acid β-oxidation cycle; depending on the metabolic state of the cell, these will either be incorporated into nascent PHA polymer chains by the PHA polymerase or will be oxidized by the β-oxidation pathway.In this study, two mutant strains of P. putida KT2442 in the PHA polymerase (phaC1) and depolymerase (phaZ) coding genes were selected. The influence of the lack of PHA accumulation abilities of this model strain has been analyzed in terms of gene regulation and central metabolism by transcriptomic and genome scale modeling approaches (3). Growth parameters such as viable cell number, PHA content, biomass, residual carbon source and released metabolites in the medium supernatants were compared in the wild type and PHA defective strains. A PHA role in balancing the stored carbon/biomass/number of individuals as function of carbon availability is demonstrated, suggesting that the presence of a PHA apparatus allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, it is demonstrated that PHA synthesis and mobilization in P. putida KT2442 is a simultaneous cycle ensuring the PHA turnover. This work opens the possibility to Systems Biology further approaches in which the prediction of fluxes in these central pathways under different external perturbations could result in the engineering of more efficient PHA production strains.1 Prieto M.A. et al., 2007. Pseudomonas V. Springer. 397-428.2 Ruth K. et al., 2008. Biomacromolecules. 9(6):1652-9.3 Nogales J. et al., 2008. BMC Syst Biol. 2:79.

P-87 Topic: Systems, Synthetic and Computational Biology

Mathematical modeling of the P. putida PTS reveals new insights into metabolite concentrations in central metabolismAndreas Kremling1, Katharina Pflüger-Grau2, Victor de Lorenzo2, Jacek Puchalka3 and Vitor Martins dos Santos3

1MPI Magdeburg, Germany, 2Centro Nacional de Biotecnología-CSIC, Spain, 3HZI Braunschweig, Germany

Pseudomonas putida possesses two distinct phosphoenolpyruvate (PEP) phosphotransferase systems. One is responsible for fructose uptake (carbohydrate branch, PTSFru) and the other is suggested to be involved in signal transduction to coordinate N and C metabolism (nitrogen branch, PTSNtr). In both branches, a high-energy phosphate from PEP is transferred by a number of proteins either to the incoming fructose or to PtsNP the last protein in the N branch. Growth of a wild type and three mutant strains of Pseudomonas putida in minimal media with different C sources (casamino acids, casamino acids with fructose and casamino acids with glucose) revealed different degree of phosphorylation of PtsNP [1]. To elucidate and to analyse the interaction between C and N metabolism, a mathematical model was set up that describe the reaction network with five reactions and 3 input parameters (concentrations of PEP and pyruvate, and fructose uptake rate). Differential equations for the components PtsP, PtsO, PtsN and FruB take into account the reaction rates for synthesis or degradation for each component. For the reaction rates, a general 2nd order mass action equation was chosen. Based on the analysis of the steady-state equation system, some of the kinetic parameters could be calculated while others have to be adjusted to describe the experimental data. Of big value were the data for PtsNP for different mutant strains. For the mutant strains, the number of state variables and the number of kinetic parameters is reduced, since some of the reactions cannot take place. As a result, all the experimental data for the wild type strain and the mutant strains could be reproduced very well with the model. The findings allow to determine the PEP/pyruvate ratio for the different environmental conditions, an important parameter, reflecting the fluxes through the central pathways of P. putida. Finally, the results were correlated with data from flux balance analysis (FBA) that determines the fluxes through central metabolism for different conditions [2]. References[1]. K. Pflueger and V. de Lorenzo. J Bac 190, pp. 3374-3380, 2008

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[2].J. Puchalka et al., PLOS Comp. Biol. 4, 2008


Mathematical modeling of the anaerobic adaptation of Pseudomonas aeruginosaRichard Münch1, Andreas Soppa2, Max Schobert2, Adam Arkin3 and Dieter Jahn2

1Technical University Braunschweig, 2Institute of Microbiology, Spielmannstr. 7, 38106 Braunschweig, GERMANY, 3Department of Bioengineering, University of California, Berkeley, USA

The anaerobic adaptation of Pseudomonas aeruginosa was analyzed and simulated via mathematical modeling. Hereby, the network was broken down into its functional modules consisting of signal transduction, gene-regulation, protein-synthesis and metabolic reactions. Kinetic and quantitative parameters were extracted from the literature or fitted with experimental data. Simulations showed a high degree of accordance with the real dynamics. Especially the adaptation of the system under fluctuating dissolved oxygen and micro-aerophilic conditions was simulated, which is the expected environment in a biofilm. It was shown using benefit-cost analyses that parts of the regulatory circuits are especially adapted to these conditions, which enabled a better insight into the design principles and the evolvement of this network.


“Omics” analysis of Medium-Chain-Length Poly-3-Hydroxyalkanoate synthesis in Pseudomonas putida KT2442 under different nutrient limitationsIgnacio Poblete Castro1, Isabel Fernandez2, Maria Auxiliadora Prieto2, Christian Jäger3, Dietmar Schomburg3, Jacek Puchalka1 and Vitor A.P. Martins dos Santos1

1Systems and Synthetic Biology Group, Helmholtz Zentrum für Infektionsforschung (HZI), Departamento de Biotecnología Ambiental, Centro de Investigaciones Biologicas/ Madrid, Spain, 3Department of Bioinformatics and Biochemistry, Technical University of Braunschweig/ Braunschweig, Germany

Pseudomonas strains, belonging to rRNA homology group I, accumulate Medium-Chain-Length Poly-3-hydroxyalkanoates (mcl-PHAs), under conditions of nutrient (N, S or P) limitation, using glucose or fatty acids as carbon source. PHAs is biopolymer, having a wide range of biomedical and industrial applications. It is mainly used as an optimal substitute for polypropylene, an essential raw material in plastic production. Huisman et al. (1992) previously highlighted that Pseudomonas putida KT2442 is able to accumulate PHA with the same composition as compared to those produced by other studied strains. However, unlike other strains, P. putida KT2442 is able to produce PHA during its exponential growth phase.This work is focused on unraveling the organization and interaction of cellular networks controlling the P. putida KT2442 PHA synthesis and its prediction by mathematical modeling. Using fatty acids as the sole carbon source, the β-oxidation pathway in P. putida KT2442 produces a pool of PHA precursors, which includes trans-2-enoyl CoA, (S)-3-hydroxyacyl CoA and 3-keto-acyl-CoA. The fadB gene encoding (S)-3-hydroxyacyl CoA dehydrogenase, one of the key enzymes involved in the last two steps of the β-oxidation pathway, was knocked out. Both the mutant and the wild type strains were then compared in parallel for PHA production using single carbon source, dual carbon-nitrogen and nitrogen limitation in a continuous culture. Preliminary results with the wild type and the fadB mutant, under carbon limitation, showed a PHA production of 20% and 40% of the CDW respectively. For both strains, the monomer composition of PHA was the same. The characterization and quantification of the PHA obtained are being carried in conjunction with several high throughput analyses involving transcriptomics, proteomics and metabolomics. This approach will allow us to adjust the PHA production in an efficient way, thus facilitating its use in various industrial applications.


Systems analysis of process-induced stresses: Unwiring the dynamic stress response of Pseudomonas putida to aromatics with time-series experimentsChristoph Ulmer1, Sarah Frank2, Christian Jäger3, Frank Schmidt4, Jens Klockgether2, Burkhard Tümmler2, Dietmar Schomburg3, Uwe Völker4, Andreas Kremling5 and Vítor Martins dos Santos6

1Helmholtz-Centre for Infection Research (HZI), 2Klinische Forschergruppe OE6711, MH Hannover, 3Department of Bioinformatics and Biochemistry, TU Braunschweig, 4Interfacultary Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt-University of Greifswald, 5Max-Planck-Institute for Complex Technical Systems, Magdeburg, 6Systems and Synthetic Biology, HZI, Braunschweig

Pseudomonas putida is an archetypal microbial cell factory: it is metabolically versatile, amenable to genetic manipulation, excellent for heterologous expression and especially tolerant to a range of physico-chemical conditions, such as solvents, hydrophobic substances, two-phase systems, low pH, low temperatures and chaotropic compounds. The exploitation of the vast potential of this organism for applications in industrial and environmental biotechnology requires a systems understanding of the metabolic and regulatory parameters that control cellular responses to process-induced stresses.We report here a Systems Biology study for the understanding of the aromatic-induced stress response in P. putida through a combination of genome-wide dynamic modelling, shock-load and time-course experiments, transcriptomics, proteomics and metabolomics. The comprehensive analyses of the data generated under those high-stress conditions has provided invaluable knowledge and enabled the generation of new hypotheses of great relevance for the production of fine chemicals by this remarkable microorganism.

Ecology and Social Microbiology

Oral presentations

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O-09 Topic: Ecology and Social Microbiology

Pseudomonas aeruginosa populations in airways of cystic fibrosis patientsSoeren Molin1, Lars Jelsbak2, Susse Kirkelund Hansen2, Lei Yang2, Martin Rau Holm2, Helle Krogh Johansen3 and Niels Hoiby3

1Technical University of Denmark (DTU), 2Dept. of Systems Biology/Technical University of Denmark, 3Dept. of Clinical Microbiology/University Hospital Copenhagen, Denmanrk

A model for the infection cycle of Pseudomonas aerginosa infections in airways of cystic fibrosis patients based on a mixture of molecular epidemiology and Systems Biology data will be presented and discussed in relation to the airway niches where P. aeruginosa is found, to the presence of other infecting microbes and to the life-style (biofilm or planctonic) of the bacteria.


Understanding lung disease in cystic fibrosis patients; the dynamics of Pseudomonas aeruginosa during infection Kenneth D Bruce1, Geraint Rogers1, Franziska Stressmann1, Thomas Daniels2 and Mary Carroll2

1King's College London, 2Southampton General Hospital, UK

Respiratory failure remains by far the most common cause of death for cystic fibrosis patients. Accordingly, it is important to understand these infections better if treatment is to be improved. Over the past decade, new culture independent approaches have been used to study respiratory samples taken from CF patients. These studies have revealed that by adulthood, a CF patient is typically colonised by a range of different bacterial species. From these initial studies, many species novel to CF lung disease, including species that require anaerobic conditions for growth, were detected. It has also been clear however that Pseudomonas aeruginosa is a common constituent of the assemblage of species present in the CF lung and is the dominant species present in c. 65% of UK adult patient respiratory samples tested. These studies offered a "snapshot" of the CF lung at a particular timepoint. Whilst informative, such studies need to be extended through a temporal analysis of species presence and absence. This has formed the basis for some more recent research that has addressed the levels of P. aeruginosa, both in absolute terms and in relation to the wider array of species present. Here again, we have taken a culture independent approach to address this question during periods of clinical stability and at times of respiratory crises. These studies, which deal with the impact of immune response and antibiotic administration, suggest new ways forward. It is hoped that studies such as this will have a beneficial longer term impact on patient health.


Social interactions and virulence of PseudomonadsAngus Buckling1

1University of Oxford

Pseudomonads perform a wide range of social interactions with their conspecifics, ranging from cooperative to antagonistic. Crucially, the nature of these interactions is likely to affect the growth rate, and hence virulence, of colonising and infecting populations. I outline theory and experiments that identify the ecological contexts in which different social behaviours evolve, and the resulting impact on virulence, and present data that suggest these results may be important in clinical situations.


PO-91 Topic: Ecology and Social Microbiology

Pseudomonas aeruginosa and its phytochrome operon: more than a photoreceptor?Katalin Barkovits1 and Nicole Frankenberg-Dinkel2

1Ruhr-University Bochum, 2Ruhr-University Bochum, Physiology of Microorganisms, Universitätsstrasse 150, 44780 Bochum, Germany

Adaptation to changing light conditions is of particular importance for phototrophic organisms and is mediated by photoreceptors. One of the first photoreceptors to be discovered in plants was a red/far-red light reversible protein called phytochrome. However, phytochromes were recently also discovered in bacteria. Whereas plant phytochromes control numerous growth and developmental processes, the biological function of bacterial phytochromes is largely unknown. The opportunistic pathogen Pseudomonas aeruginosa contains two genes coding for the necessary phytochrome components: BphO, a heme oxygenase that produces the chromophore biliverdin IX alpha and the apo-phytochrome BphP. BphP was shown to be a bonafide phytochrome in vitro. To learn more about the cellular function of this potential photoreceptor, the genetic organisation and expression was investigated. Transcriptional analyses established that bphO and bphP form a bicistronic operon. Expression of the bphOP operon was shown to be induced at increasing cell density in the stationary phase, indicating an involvement of the P. aeruginosa quorum sensing (QS) system and/or the stationary phase alternative sigma factor RpoS. While a direct involvement of the QS system could be excluded, the dependence of bphOP expression on RpoS was clearly demonstrated. Phenotypic analyses of chromosomal knock-out mutants showed no obvious growth defect under various conditions but showed decreased heat tolerance in the stationary phase indicating a potential protective role of the BphO reaction product biliverdin IX alpha. In addition, increased levels of pyocyanin and quinolones were detected in the bphO mutant strain pointing towards an additional function of BphO besides providing the chromophore for BphP. Therefore, both proteins are likely to fulfil a task in the stationary phase. Furthermore, transcriptional studies with phytochrome mutants showed a global effect on gene expression in P. aeruginosa.

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PO-92 Topic: Ecology and Social Microbiology

Conflict resolution in bacterial quorum sensing by re-evolved cooperationMartin Schuster1, Kerrigan Gilbert1 and Jessica Huie1

1Oregon State University, Corvallis, USA

In many different social systems, there is the opportunity for individuals to cheat, taking advantage of the benefits of the group, but not paying the cost of participation. We found that this is also true of communicating microorganisms (Sandoz et al. Proc. Natl. Acad. Sci. USA 2007). Bacteria communicate by sensing and responding to self-produced chemical signals, allowing the production of common goods and coordination of group activities. This mechanism is commonly referred to as quorum sensing (QS). When we evolved the model bacterium Pseudomonas aeruginosa under conditions that favor communication, signal-blind cheater variants emerged. These variants ceased production of shared extracellular factors and took advantage of their production by the group. The existence of these cheaters demonstrates the sociality of microbes, and provides a compelling resolution to the long-standing paradox in P. aeruginosa pathogenesis that although QS is required for infection in animal models, QS-deficient variants are commonly associated with infections. In addition to cheating, our evolution-in-a-test-tube experiment also revealed a mechanism of cheater control. Before cheating became detrimental to the population, a novel type of cooperator with superior fitness had evolved from a cheating ancestor. High-throughput genome sequencing of one such cheater-turned-cooperator allowed us to identify the responsible mutation, suggesting a novel regulatory mechanism of QS gene expression. Re-evolved cooperation uncovers a potential caveat with QS as an anti-virulence drug target because extracellular virulence factors can be produced even in the absence of QS. The underlying mechanism may constitute a suitable alternative target, and may provide insights into the genetic basis of transitioning from an obligate to an autonomous life style, a little explored yet important issue in evolutionary biology.

P-93 Topic: Ecology and Social Microbiology

Pseudomonas species found in asymptomatic bacterial biofilm communities on rhythm management devices differ considerably from virulent onesWolf-Rainer Abraham1, Christian Kühn2, Axel Haverich2 and Maximilian Pichlmaier2

1Helmholtz Center for Infection Research, 2Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Strasse, D-30625 Hanover, Germany

Recent work has been focused on causes of and risk factors for rhythm management device infections. The aim of this study was to elucidate whether patients may be asymptomatic carriers of bacteria on their rhythm management device, possibly allowing later manifestation of infection, and how these biofilm communities differ from infectious ones. More than 100 devices changed for battery depletion in asymptomatic patients were examined for evidence of bacterial DNA on the device and in the surrounding tissue using single strand conformation polymorphism analysis (SSCP). In 47% of the patients, bacterial DNA was demonstrated on the device, which had been in place for 64 months identified bacterial strains that are untypical for clinical device infections. The sequences identified bacterial strains that are untypical for clinical device infections. Staphylococci were demonstrated in only 3.7% of the patients and they became symptomatic within the observation interval of 2 years; all others remained asymptomatic. The known risk factors for device infections did not correlate with the demonstration of bacterial DNA in this population. Common cohabitation in the biofilm communities was identified among the strains found. The results of this study were compared with rhythm management devices explanted due to acute infections and it was found that the biofilm communities between these two types were very different. While Stenotrophomonas maltophila or Propionibacterium acnes were only detected in asymptomatic biofilms but absent in virulent biofilms, the contrary was observed for Staphylococcus aureus and Micrococcus sp. Rather high abundancies of Pseudomonas species were also detected. While P. trivialis was mostly found in asymptomatic patients, P. lini and P. flavescens occurred preferentially on infected aggregates. P. stutzeri was only detected on infected devices but the prevalence with 3% was low. P. aeruginosa was not found. Obviously, it is not essential to suppress any biofilm formation on implants but only those bacterial species characteristic for symptomatic biofilms. The role of Pseudomonas species in these biofilm infections remains to be elucidated.


Response of Pseudomonas aeruginosa PA01 to simulated microgravityAurélie Crabbé1, Rob Van Houdt2, Pieter Monsieurs2, Natalie Leys2 and Pierre Cornelis1

1Vrije Universiteit Brussel, 2Belgian Nuclear Research Centre (SCK.CEN), Boeretang 200, B-2400 Mol, Belgium

Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety, health and performance of the crewmembers. As a ubiquitous environmental bacterium, which is occasionally part of the human flora, Pseudomonas aeruginosa is found in the International Space Station. Potential changes in the behavior and virulence of this opportunistic pathogen could pose a health hazard for the immuno-compromized astronauts.Since opportunities to perform spaceflight experiments are very expensive and limited due to available crew time and safety issues, knowledge regarding the response of bacterial behavior in spaceflight conditions has improved using Earth-based models, which simulate aspects of the spaceflight environment. In this study, we used the rotating wall vessel (RWV) technology to cultivate P. aeruginosa PA01 in a low shear modeled microgravity (LSMMG) environment and we assessed the transcriptome changes with the Affymetrix GeneChip technology. One hundred thirty four genes were significantly more transcribed in LSMMG compared to the normal gravity (1g or NG) control. We identified the alternative sigma factor AlgU as well as other genes involved in the biosynthesis of the exopolysaccharide alginate, to be important in the simulated microgravity response of P. aeruginosa PA01. Accordingly, alginate was overproduced in LSMMG compared to NG, as determined with the borate-carbazole assay. Furthermore, several genes involved in the resistance to heat shock and other environmental stresses were induced in LSMMG compared to NG. We also found that transcripts of the RNA binding protein Hfq and an important part of the Hfq-regulon were more abundant in LSMMG. In line with this finding, Hfq has been previously found to play a role in the spaceflight and simulated microgravity induced virulence and -response of Salmonella enterica serovar typhimurium. In conclusion, based on transcriptomic data, several mechanisms known to be of importance for the P. aeruginosa virulence and resistance to the host defense system and antimicrobial therapies were more activated in response to the simulated microgravity environment of the RWV. Ongoing studies at the physiological level will further determine the significance of these results towards the risk for infectious disease by P. aeruginosa during spaceflight.

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Microevolution of Pseudomonas aeruginosa isolates of patients with cystic fibrosisNina Cramer1, Alessandra Bragonzi2, Moira Paroni2 and Burkhard Tümmler3

1Clinical Research Group, Hanover Medical School,Germany, 2European Institute for Cystic Fibrosis Research, Scientific Institute H.S. Raffaele, Milano, Italy , 3Clinical Research Group, Hanover Medical School, Germany

Cystic fibrosis patients typically harbour the initially acquired P. aeruginosa clone for ten years or more in their airways. In addition to genomic evolution, P. aeruginosa develops common phenotypic features irrespective of clonal descent. The conversion of phenotype and the evolution of genome organisation in P. aeruginosa isolates from CF-airways have only been studied in single, independent isolates. At Hanover Medical School there is a strain collection of sequential P. aeruginosa isolates from the onset of colonisation over a period of about 20 years. The phenotypic and genomic evolution of P. aeruginosa in CF airways was analysed in six clones each represented by sequential non-hypermutable isolates from patients with variable clinical course. In addition to phenotypic characterization first and late isolates were tested for virulence and persistence in the agar bead murine lung infection model. Growth behaviour of first and late isolates in vitro was assessed in rich, minimal and artificial sputum media in pure and mixed cultures. Sequential isolates from one patient displayed a broad spectrum of phenotypes and colony morphologies. With the exception of known phenotypes such as loss off motility or LPS conversion, there were no clear indications for gain or loss of other fitness traits during the infection course. Instead, each clone had evolved differently in its unique CF habitat. According to the mouse experiments P. aeruginosa evolved in CF airways towards a persistor pathotype attenuated in acute infection. Single growth experiments in rich and sputum media showed a higher growth rate for late than first isolates, whereas in minimal medium the first isolates were growing faster, i.e. nutrient depletion favoured the non adapted and environmentally related first isolates. In contrast late isolates were well adapted to high nutrient levels. In competitive fitness experiments one strain typically did not outcompete or influence the growth behaviour of the other strain. These experiments demonstrate that late CF isolates are not compromised in their metabolic versatility and are adapted to inhabit the atypical niche of the CF lung. The divergent evolution of the six clones in separate CF habitats points to an instrumental role of the human host in shaping the P. aeruginosa microevolution.


A new chaperone usher system in P. aeruginosaCaroline Giraud1, Marie-Cécile Lamy Lamy2, Christophe Bordi2 and Sophie de Bentzmann2

1CNRS, Marseille, France, 2CNRS LISM UPR9027 31 Chemin Joseph Aiguier 13402 Marseille cedex 20, France

The P. aeruginosa biofilm formation involves different cell surface components, like the surface appendages assembled by the chaperone-usher pathway (Cup). Several cup loci have been identified in P. aeruginosa (Vallet et a.l, 2001) and functionally characterised (Ruer et al., 2007 ; Ruer et al., 2008). We recently identified in PAO1 genome, a novel cup gene cluster, cupE, which encodes atypical pilins and until now has not been characterized. We aimed at studying CupE-dependent fimbrial assembly, implication in biofilm formation and the regulatory mechanism that controls cupE cluster expression. The CupE fimbriae implication in biofilm development was investigated through engineering deletion mutants of the different cupE genes and biofilm-dependent altered phenotypes. Confocal microscopy observation showed that several cupE gene deletions clearly attenuate the bacterial ability to structure early steps of development of the bacterial community with respect to the parental wild-type (WT) strain, phenotypes which were restored to the WT level when the mutations were complemented in cis. An antibody raised against the putative major pilin CupE1 allowed us to visualize in western blot and in transmission electron microscopy the production and assembly of the CupE pili at the P. aeruginosa cell surface. The expression level of the cupE cluster was investigated using a transcriptional fusion, cupE-lacZ, which was replaced onto the chromosome. The measurement of the β-galactosidase activity showed that this cluster is not expressed in classical laboratory culture conditions but only in biofilm formation conditions (static conditions). Thanks to a random mutagenesis strategy, we identified the two component system (TCS) PprA-PprB as an activator of cupE expression. A gel shift assay showed that the PprB-dependent activation of cupE expression is direct. This TCS seems to be a central system which controls the different components implicated in P. aeruginosa community lifestyle (Bernard et al., 2009) but also in the antibiotic resistance mechanisms (Wang et al., 2003). Thanks to CupE pili organization that ressembles the Pap or Fim pili of Escherichia coli, further studies will be dedicated to their possible interaction with eukaryotic cells.


Characterization and epidemiology of Pseudomonas syringae pv. syringae strains, causal agent of apical necrosis of mangoJose A. Gutiérrez-Barranquero1, Antonio de Vicente1, Juan C. Codina1, Alejandro Pérez-García1, Jesus Murillo2 and Francisco M. Cazorla1

1Grupo de Microbiología y Patología Vegetal, Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga. 29071-Málaga, Spain, 2Laboratorio de Patología Vegetal, ETS de Ingenieros Agrónomos, Universidad Pública de Navarra. 31006-Pamplona, Spain

Bacterial apical necrosis of mango (NAM), elicited by Pseudomonas syringae pv. syringae (Pss) limits fruit production in southern Spain and Portugal. Sprays of copper compounds (mainly Bordeaux mixture) are the typical treatment used for the control of the NAM. However, their efficacy is often limited, and it could be related to selection of copper-resistant strains of Pss. The copper-resistance genes has been cloned and characterized from Pss strains, and they are mainly associated to native plasmids. Among the plasmids described in mango strains of Pss, a 62Kb plasmid that contain homology sequences to resistance copper genes (copABCD operon) is broadly detected.In this work we performed the characterization of copper resistance genes. Hypothesis about acquisition of resistance copper determinants is supported by high diversity in these genes. In order to demonstrate this hypothesis, a study of sequence on the repA gene was performed, suggesting a possible common origin of the 62 Kb plasmids.Simultaneously, an epidemiological study of Pss strains to establish the origin and distribution of these pathogenic Pss strains is under study. Different phenotypic and genetic techniques were used to evaluate a selection of representative Pss strains isolated from mango tissues. AP-PCR (arbitrarily primed) has been used to carry out epidemiological studies. Four different primers set (ERIC1-ERIC2, BOXA1R, GTG-5 and CAG-5) were selected to analyze 125 Pss strains from different seasons and locations (including mainland Spain and Canary Islands, Portugal, Italy and Israel). To complete the study on phenotypic variability of this phytopathogenic bacterium,

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different analysis have been carried out: antibiotics resistance, copper resistance assays and dot-blot hybridization, and bioassays to determinate the production for the principal antimetabolite toxins: mangotoxin, phaseolotoxin, coronatine and tabtoxin.This research has been funded by PAI AGR169 and Proyecto Excelencia P07-AGR-2471, Junta de Andalucia, Spain.


Role of Pseudomonas in multispecies communities degrading polycyclic aromatic hydrocarbons Marcela Georgia Heck1, Thomas Neu2 and Wolf-Rainer Abraham1

1Helmholtz Center for Infection Research, Braunschweig, Germany, 2Helmholtz Center for Environmental Research, Braunschweig, Germany

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous pollutants persisting in the environment for a long time. They are very stable, highly hydrophobic and, consequently, very difficult for bacteria to degrade. However, bacteria can cooperate within a community making the degradation process viable. The aim of this work is to set-up a biofilm community able to degrade a mixture of 16 different PAHs listed as priority pollutants and to elucidate the interaction of the community members. The bacteria were obtained from soil samples of a polluted creek, a non-polluted lake and a polluted soil. Microcosms were constructed with the soil sample and sterile water and slides with the crystalline PAH mixture were exposed to it. A blank with sterile soil was used to compare the abiotic lost of PAH. Samples were taken once per week during 7 weeks and after 3 and 6 months. PAH degradation was measured with HPLC, the biofilm community diversity was determined by single strand conformation polymorphism (SSCP) of 16S rRNA gene amplicons from the total biofilm DNA. Some samples were also characterized by confocal and scanning microscopy. Bacteria were isolated from the soil and from the biofilm formed on the crystals for comparison with the taxonomic units derived from the SSCP gel and to construct a new community. The pattern of SSCP gels was different between the samples sites, indicating presence of different populations in each site, the microscopy showed always multispecies biofilms. The biofilm community from the polluted creek could degrade higher amounts of PAH than the others and was able to degrade several PAHs, including the very complex compounds benzo(a)pyrene and benzo(b)fluoranthene. This community was very diverse and comprised bacteria from the genera Aquabacterium, Bacillus and Flavobacterium and sulphur-oxidizing genera such as Thiomicrospira, Thiomonas, Rhodoferax. However, the isolates obtained both directly from the soil and the biofilm from this same sample presented completely different genera, e. g. Streptomyces, Bacillus and Pseudomonas. So far, we could prove that multispecies communities are a promising alternative for the control of PAH pollution, especially those that were already exposed to pollutants. New communities are now constructed with different isolates from this sample including Pseudomonas, that we believe, will play an important role in the degradation due to the ability of this genus to degrade many pollutants.



LapF, the second largest Pseudomonas putida protein, is involved in plant surface colonization and biofilm developmentMarta Martínez-Gil1, Fátima Yousef-Coronado1 and Manuel Espinosa-Urgel1

1Estación Experimental del Zaidín. CSIC, Granada, Spain

Bacterial populations frequently appear forming multicellular communities associated with solid surfaces, known as biofilms. Although these communities are present in abiotic surfaces, bacteria often attach to and colonize biotic surfaces. We have been studying molecular and genetic determinants specifically involved in the adhesion of microbial cells to plant surfaces (seeds and roots), as a possible previous step to the establishment of biofilm-like structures. In this work we have used as a model P. putida KT2440, an efficient colonizer of the rhizosphere (root surface and surrounding soil area) of many plant species. It is broadly believed that proteins localized in the bacterial surface play an important role in cell-cell interactions and adhesion to different surfaces. Previous work allowed the characterization of LapA, the largest protein of P. putida KT2440 (8682 amino acids), essential for adhesion to seeds, root colonization and biofilm formation on abiotic surfaces. We are currently characterizing a second large protein implicated in adhesion processes, which we have called LapF. LapF is 6310 amino acids long, with 3 different domains, belonging to a family of large repetitive proteins closely related to LapA. One of them includes 63 imperfect repeats of 85-90 amino acids, and the C-terminal domain shows calcium binding sites and a potential secretion domain. Calcium promotes adhesion of P. putida cells to solid surfaces. A mutant in lapF with a truncated C-terminal domain presents reduced capacity to attach to seeds and is affected in competitive rhizosphere colonization. Whereas biofilm formation in static conditions is not significantly different between the wild type and this lapF mutant, the development of a mature biofilm is impaired in the mutant under flow conditions. Using specific antibodies against LapF, its specific localization in the cell has been analyzed by Western blot. LapF is exported to the cell surface and the C-terminal region seems to be essential for its secretion to the surface. It is worth noting that a potential operon encoding the elements of an ABC transporter is found adjacent to the lapF gene.Our results suggest LapF may be a mediator between P. putida KT2440 and plant surfaces and could play a role in cell-cell interactions.


Regulation of motility in P. fluorescens through the Gac signalling pathwayFrancisco Martinez-Granero1, Ana Navazo1, Emma Barahona1, Miguel Redondo-Nieto1, Marta Martin1 and Rafael Rivilla1


In many Gram-negative bacteria, the GacA/GacS system positively controls the expression of secondary metabolites and exoenzymes required both for virulence and for biocontrol. This regulatory cascade functions at a posttranscriptional level involving RNA-binding proteins as a key regulatory element. We have previously shown that in the biocontrol agent P. fluorescens F113 swimming motility is

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negatively regulated by the Gac system. Here, it is shown that the same regulation occurs in the model strain P. fluorescens Pf0-1. Overexpression of either RNA-binding protein RsmA or RsmE mimicked the phenotype of a gac mutant, indicating that positive (secondary metabolites) and negative (motility) regulations occur through the same pathway. The enhanced motility phenotype showed by both gac mutants and Rsm-overexpressing strains, is due to an increase in the level of transcription of the fleQ and fliC genes. Since Gac regulation through the Rsm pathway takes place at translational level (blocking RNA translation), for negative regulation of motility the RNA blocked should encode a repressor of fleQ transcription. We have tested several genes that encode proteins that have been previously shown to repress fleQ expression in several pseudomonads: Vfr, MorA and AmrZ. A mutation in the vfr gene had no effect on swimming motility in P. fluorescens. A morA mutant surprisingly showed reduced swimming motility, indicating that conversely to Pseudomonas putida, MorA is a motility activator protein in P. fluorescens. An amrZ mutant showed enhanced motility when compared to wild-type strain. Moreover, no significant differences in swimming motility were observed between the gac mutant and the gac-amrZ double mutant indicating that both belong to the same motility repression pathway.


Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle Heidi Mulcahy1 and Shawn Lewenza1

1University of Calgary, Canada

The ability of Pseudomonas aeruginosa to cause a range of diseases in humans is due, in part, to its capacity to regulate gene expression in response to specific environmental conditions. The biofilm mode of growth is thought to contribute to the ability of P. aeruginosa to persist long-term in susceptible hosts and cause chronic infections.In this study we tested the hypothesis that the biofilm lifestyle is brought about by specific signal transduction cascades in response to distinct environmental signals from the host, leading to long-term survival of P. aeruginosa in the lungs of Cystic Fibrosis (CF) patients. A panel of 22 plasmid or chromosome-encoded transcriptional lux fusions to crucial two component system (TCS) genes, biofilm adhesins and matrix exopolysaccharides (EPS) were analysed in high throughput gene expression profiling assays in 40 different media types, intended to mimic the challenges faced during chronic lung infections. Here we describe the identification of magnesium (Mg2+) limitation as a relevant in vivo signal that alters the expression of key regulatory and structural biofilm-associated genes and induces biofilm formation in P. aeruginosa. Low Mg2+ promoted aggregation and biofilm formation by positive regulation of the EPS biosynthetic operons pel and psl. In addition, low Mg2+ repressed motility, as well as type II and type III secretion system genes. Furthermore, Mg2+ limitation transcriptionally repressed the expression of retS while inducing ladS, gacS, gacA and expression of the regulatory RNAs, rsmZ, rsmY and rgsA.We have recently demonstrated that extracellular DNA is a cation chelator indicating that DNA-rich environments such as biofilms and the CF lung are likely cation- and Mg2+-limited (1). In P. aeruginosa, only 35% of the response to Mg2+ limitation is estimated to occur through the PhoPQ or PmrAB TCSs (2), suggesting that additional regulators are involved. Our data indicates that RetS, LadS and GacS play a role in the Mg2+ response, in a direct or indirect manner. The identification of low Mg2+ as a relevant in vivo signal that promotes biofilm formation, induces antibiotic resistance and immune response evasion is the first demonstration of a specific environmental signal regulating the genes that are predicted to control the switch from acute to chronic infection in P. aeruginosa. (1) Mulcahy et al., 2008. PLoS Pathog. Nov;4(11):e1000213.(2) McPhee et al., 2006. J Bacteriol. Jun;188(11) 3995-4006.


The flagella-specific σ-factor FliA co-ordinates expression of the Aer2 energy-sensor with motility in Pseudomonas putidaSofia Österberg1 and Victoria Shingler1

1Umeå University, Spain

Soil dwelling Pseudomonas putida strains have a versatile metabolism that is frequently augmented by auxiliary pathways carried on plasmids. The strain CF600, for example, harbours a plasmid that allows the bacterium to degrade the toxic aromatic compounds phenol and methylated phenols. For efficient degradation and removal of pollution by such potentially useful strains, the bacteria need to both sense and move towards the pollutant and optimal O2 conditions for its catabolism. Using motility plate assays and specific mutants, P. putida CF600 and KT2440 have been shown to exhibit taxis towards the aromatic compounds that they can catabolise and not towards other aromatics. Taxis towards (methyl)phenols is thus metabolism-dependent. Mutational analysis revealed that Aer2 - the most abundant of three differentially regulated Aer-like receptors of KT2440 - is responsible for taxis towards phenolics and other readily metabolised compounds and aerotaxis through O2 gradients (1). Since true redundancy in bacteria is rare, we were interested in determining the mechanism underlying differential expression of the three Aer-like receptors. Here we show that in contrast to the promoters for aer1 and aer3, the promoter for aer2 is controlled by the FliA-RNA polymerase (FliA-RNAP). FliA is a specialized sigma factor involved in expression of the flagellum genes that are hence required for bacterial taxis-based motility. We will present our analysis that demonstrate that transcription of aer2 is severely decreased in strains lacking FliA, and how in vitro transcription is modified through mutation of key signatures of the aer2 promoter. Through Aer2, P. putida has the ability to sense different energy sources without having a specialized receptor that recognizes each specific compound. Our finding that expression of this versatile receptor is coupled to expression of the motility apparatus via FliA suggests that this signalling system contributes to the environmental success and metabolic versatility of P. putida.1. Sarand, et al. (2008) Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida. Environ. Microbiology. 10:1320.


Ca2+-binding protein CasA increases P. aeruginosa fitness to a host environmentMarianna A Patrauchan1, Svetlana Sarkisova2 and Michael Franklin3

1Oklahoma State University, 2Johnson Space Center, 3Montana State University, USA

Calcium (Ca2+) regulates essential processes in eukaryotes. Even a slight imbalance in Ca2+ homeostasis may cause cell dysfunction and disease. Ca2+ is a part of a hyperinflamatory host response to bacterial infection, and accumulates in airway epithelia and pulmonary liquids of cystic fibrosis (CF) patients. CF patients are particularly susceptible to infections with P. aeruginosa, which forms biofilms on

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airway mucosal epithelium, causing airway blockage and death. The regulatory role of Ca2+ in bacterial pathogenicity has not been demonstrated. Our research focuses on the interplay between Ca2+ and P. aeruginosa pathogenicity.To elucidate Ca2+-signal transduction pathways, we performed bioinformatic prediction of Ca2+-binding proteins in the P. aeruginosa genome. Based on 44% sequence identity with calsymin (CasA, YP_472788) and presence of EF-hand domains with Ca2+-binding loops, we predicted that the hypothetical protein encoded by PA4107 is Ca2+-binding. We designated it CasA and hypothesized that it is involved in Ca2+-signaling. The deletion mutant lacking casA was generated and functional studies were performed. A global quantitative proteomic analysis revealed significant changes in the mutant’s protein profiles. Importantly, the deletion had an affect only in the cells grown at elevated [Ca2+]. Among the proteins that were significantly less abundant or absent in the mutant, we identified a trigger factor (PA1800), whose gene is tightly clustered with clp proteases, and stress related proteins: heat shock protein HtpG (PA1596), universal stress protein UspA (PA4352), and a hypothetical protein PA1069, whose gene is closely clustered with a heat shock protein (PA1068). We also identified a phenazine biosynthesis protein PhzD (PA4213), an acyl-carrier-protein involved in fatty acid metabolism FabG (PA2967), and a hypothetical protein PA3329, predicted to be involved in siderophore biosynthesis. Previously, we showed some of these proteins were induced by elevated [Ca2+] in P. aeruginosa FRD1 biofilms. Thus, deletion of casA modifies P. aeruginosa FRD1 response to elevated Ca2+. Most of these changes are directed towards increasing bacterial resistance to stress conditions and inducing production of virulence factors. Such changes may be beneficial for P. aeruginosa growth in a host environment. We are currently determining Ca2+ homeostasis in P. aeruginosa, which is important to study the signaling role of Ca2+ in P. aeruginosa pathogenicity.


Antagonistic activity of Pseudomonas fluorescens SWRI196 against the olive tree pathogen Pseudomonas savastanoi Hassan Rokni Zadeh1 and René De Mot2

1Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, , Heverlee, Belgium, 2Centre of Microbial and Plant Genetics, K.U.Leuven, Heverlee, Belgium

Olive knot caused by Pseudomonas savastanoi is the main bacterial disease of olive plants and chemical bactericides are not economically beneficial to control it. Moreover, development of resistance and environmental concerns force to employ alternative, more selective and environmental friendly control methods such as biological control. Diverse biomolecules that mediate antagonism among plant-associated bacteria provide a potential resource for novel compounds.A collection of about 320 fluorescent Pseudomonas strains isolated from wheat, maize, rice and banana rhizospheres in Iran, Belgium, France and Sri Lanka were screened against fourteen strains of P. savastanoi, using a diffusion assay in trypticase soy agar. To our knowledge this is the first study to explore the potential of in vitro P. savastanoi antagonism for a collection of phylogenetically more distant non-pathogenic Pseudomonas isolates from different plant rhizospheres and geographical origins.Strain SWRI196 showed inhibitory activity against all tested P. savastanoi strains, as well as most other Pseudomonas species tested, including Pseudomonas aeruginosa. Growth inhibition was also observed for several other proteobacterial species, representative Gram-positive bacteria, and Candida albicans. Microbiological characterization and 16S rDNA sequence analysis identified the strain SWRI196 as P. fluorescens. The antibacterial activity of strain SWRI196 was not affected by protease or heat treatment indicating a non-proteinaceous nature of the active compound(s). A combined approach is used to characterize the biosynthesis and chemical nature of the compound(s). In order to identify genes required for production, a mutant library of P. fluorescens SWRI196 was constructed using plasposon pTnMod-OKm'. Several null mutants have been identified and the genes affected are being characterized by sequence analysis of the respective transposon insertion sites.


Behaviors of the IncP-7 carbazole degradative plasmid pCAR1 and its host genus Pseudomonas strains in the artificial environmental samplesMasaki Shintani1, Hisakazu Yamane1 and Hideaki Nojiri1

1Biotechnology Research Center, The University of Tokyo, Japan

Bioaugmentation is a method to remove pollutants from the contaminated sites by inoculating contaminant-degrading bacteria into the site. Inoculation of a bacterium carrying a conjugative degradative plasmid would enable to prevent the loss of the degradative genes from the site once they were transferred into indigenous bacteria. However, in contrast to laboratory conditions, knowledge of the behavior of such plasmids (or hosts) in the natural condition is insufficient to predict the fate of the plasmid and its hosts in the environment. Incompatibility (Inc) group P-7 plasmid pCAR1 is a conjugative and carbazole degradative plasmid, whose features for replication, partition, and transfer have been investigated in our laboratory. To analyze the behaviors of pCAR1 and its hosts in the environment, we introduced a simple detection system for conjugative transfer using pCAR1 tagged with a transfer reporter gene encoding red fluorescent protein (pCAR1::rfp) (1). We also constructed artificial samples of the natural environment using sterile soil and river or pond water to obtain high reproducibility (2). As inoculated strains, we used P. putida KT2440, P. fluorescens Pf0-1, P. chlororaphis IAM1511, and P. resinovorans CA10dm4 (the host of pCAR1::rfp), and other plasmid-free Pseudomonas and non-Pseudomonas strains. Then the behaviors of pCAR1 and its host strains and other strains in these artificial samples were monitored. The results were summarized as follows; (i) KT2440 was the most appropriate host strain to degrade carbazole in the artificial soil with the high water contents, (ii) CA10dm4 was the best in the artificial environmental water, (iii) The frequency of pCAR1 transfer in the artificial soil are below the detection limit with every host strain, (iv) the presence of Ca2+ and Mg2+ are necessary for pCAR1 to transfer from KT2440 in the artificial water. Interestingly, we found some Pf0-1 strains improved their degradability for carbazole after inoculation into artificial pond water. Now we are analyzing the reappeared Pf0-1L(pCAR1::rfp) to understand why they were able to degrade carbazole faster than the original one. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences in Japan. (1) Shintani et al., 2008, Biotechnol Lett 30:117-122. (2) Shintani et al., 2008, Appl Microbiol Biotechnol 80:485-497.


Within-patient diversity of Pseudomonas aeruginosa quorum sensing populations from cystic fibrosis infectionsCara N Wilder1, Gopal Allada2 and Martin Schuster1

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1Oregon State University, 2Oregon Health & Science University, Corvallis, USA

In the opportunistic pathogen Pseudomonas aeruginosa, acyl-homoserine lactone quorum sensing (acyl-HSL QS) regulates the expression of hundreds of genes, many of which are required for virulence. Paradoxically, QS-deficient variants, often carrying mutations in the central QS regulator lasR, are frequently isolated from infections, including cystic fibrosis (CF) lung infections. The proportion of QS-deficient variants in individual infections is not known. Based on recent in vitro evidence we hypothesized that these QS mutants emerge as social cheaters that take advantage of extracellular factors produced by the QS-proficient majority, suggesting that mutants arise because, not despite, QS is important during infection. If QS is important for chronic CF lung persistence, then QS-deficient variants should only comprise a small fraction of the P. aeruginosa population in an infected individual. To test this prediction, we assayed a panel of 135 P. aeruginosa isolates from eight different adult CF patients (9-20 isolates per patient) for diverse QS-controlled phenotypes. In most cases, we found mixtures of partially and fully QS-deficient isolates. Acyl-HSL, sequencing and complementation analysis of variants with global loss-of-function phenotypes revealed dependency upon central QS circuitry genes lasR, lasI, and rhlI. Mutant and wild type isolates were clonally related, implying that QS-deficient variants evolved from wild type ancestors in vivo. The low QS activity of P. aeruginosa populations in many CF sputum samples questions the general significance of QS and hence, social exploitation, in advanced CF lung infection, and raises doubt about the suitability of QS as a novel anti-virulence drug target.


Interactions in mixed-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus Liang Yang1, Niels Hoiby2 and Tim Tolker-Nielsen2

1Technical University of Denmark, 2University of Copenhagen, Denmark

Microbial lung infection is the leading cause of morbidity and motality in cystic fibrosis (CF) patients. The lungs of chronically colonized CF airways represent a complex ecosystem in which the interspecies interactions of different organisms remain largely unelucidated. It is believed that failure of eradication of CF infections is largely due to the formation of bacterial biofilms, which are complex bacterial communities consisting of microcolonies embedded in self-produced extracellular polymer substances (EPS). Pseudomonas aeruginosa and Staphylococcus aureus are two of the major species that colonize CF airways, and they are notorious for their tolerance towards antibiotic treatments due to their ability to form biofilms. Here, we have investigated interactions of P. aeruginosa and S. aureus - when grown in co-culture biofilms. By growing mixed-species biofilms of P. aeruginosa and S. aureus in the standard flow chamber system and observing them with confocal laser scanning microscopy, we show that P. aeruginosa and S. aureus associate with each other and form two-species microcolonies. We found that P. aeruginosa type IV pili are necessary for its association with S. aureus. In addition, DNase treatment experiments suggested that extracellular DNA is involved in the type IV pili mediated association of P. aeruginosa and S. aureus in coculture biofilms in accordance with clinical experience of DNase treatment (Acta Paediatric. Scand. 95:1070; 2006). Finally, we found that two-species microcolonies in co-culture biofilms could increase tobramycin resistance of both P. aeruginosa and S. aureus cells.

Cell-to-Cell Communication and Intracellular Signalling

Oral presentations

O-12 Topic: Cell-to-Cell Communication and Intracellular Signalling

The RSCV phenotype of P. aeruginosaMatthew R Parsek1

1University of Washington, USA

Laboratory biofilm growth and chronic CF infections give rise to the rugose small colony variants. These variants are characterized by elevated levels of cyclic-di-GMP and overexpression of the Pel and Psl polysaccharides. These variants exhibit several characteristics that might contribute to persistence in the CF environment, including elevated biofilm formation, aminoglycoside resistance and reduced stimulation of host inflammatory chemokine signaling. One genetic route to this phenotype involves mutations in the wspF gene, resulting in activation of the GGDEF diguanylate cyclase, WspR. However, we provide evidence that at least two other classes of mutations produce this phenotype. A random transposon mutagenesis screen of a wspR mutant strain produced multiple insertions conferring the RSCV phenotype. An analysis of these transconjugates revealed mutations in the RsmA signaling pathway and flagellar biosynthesis genes induce the RSCV phenotype. Collectively, our analysis revealed new relationships between cyclic-di-GMP signaling, the RsmA regulatory pathway, motility and the RSCV phenotype.

O-13 Topic: Cell-to-Cell Communication and Intracellular Signalling

Characterization of starvation-induced dispersion in Pseudomonas putida biofilmsTim Tolker-Nielsen1, Morten Gjermansen2, Martin Nilsson1 and Liang Yang3

1University of Copenhagen. Faculty of Health Sciences., 2Novozymes A/S. Bagsvaerd, Denmark., 3Department of Systems Biology. Technical University of Denmark.

The biofilm developmental cycle comprises both biofilm formation and biofilm dispersal processes. Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene lapF. Sequence and structural analysis has suggested that the LapF geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface proteins. We provide evidence that the putative cysteine proteinase LapF is involved in P. putida OUS82 biofilm dispersal through modification of the outer-membrane-associated protein LapA. The biofilm dispersal defect of the P. putida lapF mutant is reflected in a high cell-to-cell adhesiveness and hydrophobicity of the cells, which can be decreased to the low hydrophobicity level of planktonic wild type cells by

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proteinase K treatment, suggesting that it is caused by membrane-associated protein, and in agreement with the predicted function of the LapF protein. Transposon mutagenesis, screening, and mutant characterization provided evidence that the outer-membrane-associated protein LapA is causing the high hydrophobicity of the P. putida lapF mutant. While the P. putida lapF mutant formed more biofilm than the wild type, P. putida lapA and P. putida lapAF mutants displayed decreased surface adhesion and were deficient in subsequent biofilm formation, suggesting that LapF affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c-di-GMP level via induction of an EAL domain protein led to dispersal of P. putida wild type biofilm but did not disperse P. putida lapF biofilm, indicating that LapF exerts its activity on LapA in response to a decrease in the intracellular c-di-GMP level.


PO-109 Topic: Cell-to-Cell Communication and Intracellular Signalling

Cell density dependent regulation in Pseudomonas putida KT2440Regina Fernández-Piñar1, Juan Luis Ramos2, Stephan Heeb3, Miguel Cámara3 and Manuel Espinosa-Urgel2

1Estación Experimental del Zaidín. CSIC, 2Estación Experimental del Zaidín. CSIC. , 3Centre for Biomolecular Sciences. University of Nottingham. Nottingham, United Kingdom

Many microbial species are known to produce small diffusible molecules, termed autoinducers, which mediate cell-cell communication in a population density-dependent manner, inducing changes in gene expression when a certain concentration of the signal molecule is reached. This signalling mechanism is known as quorum sensing (QS). Acyl-homoserine lactones (HSLs), the major quorum sensing signals, are the products of LuxI-type autoinducer synthases and are detected by cognate cytoplasmic LuxR proteins that bind DNA and activate transcription of target quorum sensing genes. We are attempting to elucidate the QS-regulated signalling mechanism in Pseudomonas putida KT2440, an efficient root-colonizing bacterium that survives in the plant rhizosphere at high cell densities. Its genome lacks luxI homologs; consistently, our data on the profile of HSLs molecules produced by KT2440 reveal that only trace amounts of C4-HSL and C12-HSL can be detected. Limited production of other known QS signals, namely quinolones, can also be found. However, we have previously characterized a gene, ddcA, involved in colonization of plant seeds, which shows an expression pattern dependent on cell density, which suggests the presence of an intraspecific QS signal in this strain yet to be defined. We have also found that ddcA expression responds to signals accumulated in the supernatant of other Pseudomonas species cultures. In a search for ddcA regulators we identified a two-component regulatory system referred to as RoxS/RoxR. The genes coding for RoxS and RoxR form an operon and a mutant in roxSR is affected in cytochrome oxidase activity and shows reduced fitness in the rhizosphere of corn plants. A global transcriptional analysis using microarrays was performed to unravel the RoxS/R regulon. Results show that certain amino acid metabolic enzymes, surface structures, cytochrome c, cytochrome oxidases and electron transport systems, among others, are under the influence of RoxS/R. Our data suggest an overlap between redox state and cell density dependent regulation.


How do RsmA/CsrA-like proteins specifically recognise their mRNA targets in Pseudomonas fluorescens CHA0?Karine Lapouge1 and Dieter Haas2

1University of Lausanne, 2University of Lausanne, Department of Fundamental Microbiology, Lausanne, Switzerland

The rhizosphere bacterium, Pseudomonas fluorescens CHA0, has a sophisticated system protecting the roots of host plants against pathogenic fungi by synthesizing diverse extracellular products such as antibiotics, exoproteases and hydrogen cyanide. The biosynthesis of these biocontrol compounds is strictly regulated by a quorum sensing two-component system, GacS/GacA, which induces the transcription of three small noncoding regulators RNAs (RsmX, RsmY, RsmZ). These capture the translational repressor proteins RsmA and RsmE. In previous work we have shown that the RsmA/E proteins repress the translation of hcnABC mRNA (encoding hydrogen cyanide synthase) by binding to an extended GGA motif in the ribosome binding site (RBS) as well as to GGA motifs further upstream. In particular, the interaction of the RsmA/E proteins with the RBS of the hcnA gene promotes the formation of a stem-loop structure. This binding hinders ribosome access and translation initiation. The resulting repression is prevented when the sRNAs with repeated unpaired GGA motifs titrate the RsmA/E proteins. To refine this recognition model, we have investigated the binding specificity of RsmA/E proteins for mRNAs of various biocontrol compounds that are expressed under GacA control. By the use of translational lacZ fusions to the phlACBD genes (encoding 2,4-diacetylphloroglucinol biosynthetic enzymes) and the pltLABCDEFG genes (encoding pyoluteorin biosynthetic enzymes), we show that the RBS sequence of these genes is involved in Gac/Rsm regulation and that the stem-loop structures encompassing the RBS can be variable.


Gene regulation in Pseudomonas: from environmental signals to responses via global post-transcription control and intracellular messagingMarco Messina1, Karima Righetti1, Woo Jin Lim1, Stephan Heeb1, Paul Williams1 and Miguel Cámara1

1University of Nottingham, UK

In Pseudomonas aeruginosa PAO1 the global post-transcriptional regulator RsmA together with the regulatory RNAs RsmY and RsmZ regulate the translation rates of many virulence-related genes, including the N acyl-homoserine lactone synthase genes lasI and rhlI and the genes required for hydrogen cyanide, lectin, and rhamnolipid biosynthesis. A functional RsmA protein is also required for swarming, and as we previously discovered that overexpressing c-di-GMP phosphodiesterases can restore swarming motility in an rsmA mutant, we searched for additional components mediating this signal transduction in relation with global post-transcriptional control and bacterial adaptation to the environment. Screening of Tn5 mutants and of a genomic library for restoration of swarming in an rsmA mutant revealed that several genes, either when overexpressed or knocked out, can re-establish this motility. While the genes identified by transposon mutagenesis were of diverse functions, the majority of those found by screening the genomic library encoded proteins containing both GGDEF and EAL domains likely to be c-di-GMP phosphodiesterases. This suggests that increased c-di-GMP levels are

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in part responsible for preventing the swarming in the rsmA mutant. In parallel, a Tn5 mutagenesis on a the wild type PAO1 strain carrying a chromosomally encoded rsmZ-lux fusion revealed that disruption of wspF, encoding a methyl esterase that negatively controls the activity of the WspR c-di-GMP cyclase, highly induced the rsmZ promoter. This enhanced activity confirmed by an in-frame deletion of wspF could in turn be reduced to wild type levels by overexpressing the c-di-GMP phosphodiesterases previously found to restore swarming in the rsmA mutant, indicating that rsmZ expression follows c-di-GMP levels. Finally, an in-frame deletion of the gene encoding the c-di-GMP binding protein PilZ caused a strong activation of the chromosomal rsmZ-lux reporter fusion, suggesting that c-di-GMP mediates a derepression of the rsmZ promoter via PilZ. Taken together this indicates the existence of an important and sophisticated intracellular network involving the secondary messenger c-di-GMP combined with the Rsm global post-transcriptional regulation system.


Three independent signalling pathways repress motility in Pseudomonas fluorescens F113Ana Navazo1, Emma Barahona1, Miguel Redondo-Nieto1, Francisco Martinez-Granero1, Rafael Rivilla1 and Marta Martin1


Motility is one of the most important traits for rhizosphere colonization by Pseudomonas. Despite this importance, motility is severely repressed in the rhizosphere colonizing strain Pseudomonas fluorescens F113. This bacterium, which has applications in biocontrol and rhizoremediation, is unable to swarm under laboratory conditions and produce relatively small swimming haloes. However, phenotypic variants with the ability to swarm and producing swimming haloes up to 300% larger than the wild-type strain, arise during rhizosphere colonization. These variants harbour mutations in the genes encoding the GacA/GacS two component system and in other genes. In order to identify genes and pathways implicated in motility repression, we have used generalized mutagenesis with transposons. Analysis of the mutants has shown that besides the Gac system, the Wsp system and the sadB gene, which have been previously implicated in cyclic-di-GMP turnover, are implicated in motility repression: mutants in the gacS, sadB, or wspR genes can swarm and produce swimming haloes larger than the wild-type strain. Epistasis analysis has shown that the pathways defined by each of these genes are independent, since double and triple mutants show an additive phenotype. Furthermore, GacS, SadB and WspR act at different levels. Expression of the fleQ gene, encoding the master regulator of flagella synthesis is higher in the gacS- and sadB- backgrounds than in the wild-type strain and this differential expression is reflected by a higher secretion of the flagellin protein FliC. Conversely, no differences in fleQ expression or FliC secretion were observed between the wild-type strain and the wspR- mutant.

P-113 Topic: Cell-to-Cell Communication and Intracellular Signalling

Energy sensing and cyclic di-GMP signalling in P. putidaAnna Åberg1, Karina Herrera Seitz2, Sofia Österberg1 and Victoria Shingler1

1Umeå University, Sweden, 2University Nacional de Mar del Plata, Buenos Aires, Argentina

Bacteria use extensive regulatory networks to co-ordinate their physiology to prevailing nutritional and stress conditions. One of the most recently identified, and least understood, involves the nucleotide second messenger cyclic di-GMP (c-di-GMP). This near ubiquitous bacterial signalling molecule has profound effects on numerous bacterial life-style and adaptive process, but in many cases mechanistic information is lacking. c-di-GMP turn-over is mediated by GGDEF- and EAL-domain proteins that mediate opposing diguanylate cyclase and phosphodiesterase activities, respectively. Bacterial genome sequences have shown that GGDEF- and EAL-domains are highly abundant, widely distributed, and often occur within the same protein. However, in most dual GGDEF-EAL-domain proteins only one of the two domains is catalytically active, with the other domain adapted to serve a regulatory role.The experimental system under scrutiny involves the Pseudomonas putida KT2440 derived PP2258 protein that is encoded in a bi-cistronic operon along with the energy sensor Aer1. PP2258 is a tri-domain (PAS-GGDEF-EAL) protein that appears to be one of the rare examples of a c-di-GMP turnover protein in which both catalytic domains are active. Alanine substitutions within the PP2258-SGDEF motif reduce c-di-GMP production, whereas an alanine substitution within the PP2258-EGL domain resulted in > 7-fold increase in c-di-GMP levels as measured by two-dimensional thin layer chromatography. Within the bacterial cell, PP2258 is involved in regulating motility of P. putida since a PP2258 null mutant is defective in motility on plate. However, electron microscopic analysis has revealed that the null mutant possesses the same number of flagella in its polar bundles as the wild-type strain. This suggests that PP2258 regulates motility via c-di-GMP signalling to an adaptor protein, rather than regulating the expression of the flagella components. Possession of a PAS domain combined with co-regulation with an energy-sensor further implicates PP2258 and c-di-GMP signalling in coupling control of motility to the energy status of the cell. We will present our most recent mutagenesis analysis of the PP2258 protein aimed to test this idea.


NtrC- mediated activation of sigma 54 promoters in Pseudomonas putida Ines Canosa1, Ana B. Hervas1, Richard Little2, Ray Dixon2 and Eduardo Santero1

1University Pablo de Olavide/CSIC, Sevilla, Spain, 2John Innes Centre, Norwich, UK

Nitrogen regulatory protein (NtrC) in Pseudomonas putida activates transcription of genes/operons that promote transport and/or assimilation of alternative nitrogen sources under nitrogen-limiting conditions. The nitrogen regulatory cascade in P. putida comprises several signal transduction proteins such as glutamine synthetase/glutamate synthase (GS/GOGAT), uridylyltransferase (UR/UTase) and PII protein. The only PII protein found in P. putida is GlnK and is encoded by glnK gene. We have examined the NtrC- activated expression pattern of glnK and defined the sequence elements determining the binding of the sigma 54- containing RNA polymerase to the glnK promoter. We show that, contrary to enterobacteria, activation of glnK promoter is greatly dependent on the presence of the integration host factor IHF both in vivo and in vitro. We have also studied three other operons that in enterobacteria are NtrC controlled but its activation is mediated by the regulatory protein Nac, who acts as a linker between NtrC and sigma 70 RNA polymerase -dependent promoters. We show that in P. putida, their homologues are directly activated by NtrC.


Oligomerization of the H-NS family member MvaT is important for DNA-bindingSandra Castang1 and Simon L. Dove2

1Children's Hospital Boston - Harvard University, 2Children's Hospital Boston - Harvard University, USA

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MvaT is an abundant DNA-binding protein that controls the expression of a large number of genes in Pseudomonas aeruginosa. Despite limited sequence similarity, multiple functional similarities and several structural predictions suggest that MvaT belongs to the H-NS family of nucleoid-associated proteins, members of which typically function by repressing transcription. H-NS consists of two functionally different domains separated by a flexible linker. The C-terminal domain is involved in DNA-binding while the N-terminal domain and linker are required for oligomerization. H-NS binds to the DNA as at least a dimer and is thought to polymerize across adjacent AT-rich regions of DNA. Because the ability of H-NS to oligomerize across the DNA is thought to be essential for its biological functions we investigated the in vivo quaternary structure of MvaT and mapped the regions responsible for these interactions using a combination of bacterial two-hybrid and one-hybrid assays. We present evidence that MvaT can form both dimers and higher order oligomers, and that the N-terminal region of MvaT is required to mediate both dimer and higher-order oligomer formation. We also report the identification of amino acid residues in the N-terminal region of MvaT that appear to be important for higher-order oligomer formation but not for dimer formation. Using electrophoretic mobility shift assays and chromatin immunoprecipitation we show that MvaT mutants that are defective for higher-order oligomer formation are defective for DNA-binding in vitro and in vivo. Our findings support a model in which protein-protein interactions between adjacently bound dimers of MvaT are critical for DNA-binding and suggest that the ability to form higher-order oligomers is a conserved and essential feature of H-NS family members.


A phage protein (SipA) involved in superinfection exclusion identified from Pseudomonas aeruginosa phage D3112You-Hee Cho1, Yun-Jeong Heo1 and Hyun-Hee Jung1


Phage infection-derived lyogenic conversion has profound effects on bacterial physiology. We previously observed the superinfection exclusion (SIE) phenomenon between Pseudomonas aeruginosa phages (Heo et al. 2007. Microbiol. 153: 2885-2895). Here we determined SIE phenotypes between D3112, MP22, and DMS3 as well as three newly isolated P. aeruginosa phages, MP29, MP42 and MP45, which belong to the same family (Siphoviridae). The six phages were divided into two subgroups (MP29 subgroup: D3112, MP29, MP42 vs. MP22 subgroup: MP22, MP45, DMS3) based on the c repressor homology, and the superinfection exclusion was observed between the phages belonging to the same subgroup, except for D3112 whose lysogen excluded the superinfection by all the phages. Furthermore, D3112 lysogen was completely defective for type IV pilus (TFP)-mediated twitching motility, although the phage adsorption was not affected at all. We searched for D3112-specific gene(s) based on the comparison of whole genome sequences of D3112, MP22 and DMS3. As a result, we found that the mutation of a region unique to D3112 (R2) restored the twitching motility defect and SIE phenotypes of D3112 lysogen. Furthermore, a multicopy, but not a single copy-based expression of D3112 ORF5 (SipA, superinfection in Pseudomonas phage) in the R2 region was sufficient for superinfection exclusion, but not for twitching motility defect. These results suggest that SipA is involved in superinfection exclusion, presumably by modulating TFP retraction, which is sufficient for phage uptake but requires additional gene(s) for twitching motility modulation.


Post-transcriptional regulation of anthranilate metabolism in Pseudomonas aeruginosa by acyl-homoserine lactonesSudha Chugani1 and E. Peter Greenberg1

1University of Washington, Seattle, USA

Bacteria employ a mechanism of cell-cell communication called quorum sensing to coordinately regulate gene expression in response to alterations in population density. Quorum-sensing systems similar to the archetypical LuxI/R system of Vibrio fischeri have been found in over 50 bacterial species where they function as regulators of a range of phenotypes. In Pseudomonas aeruginosa quorum control of gene expression is affected by three LuxR-type signal receptors LasR, RhlR, and QscR in response to the acyl-homoserine lactone (acyl-HSL) signals 3OC12-HSL and C4-HSL. Transcriptome analyses aimed at exploring the universality of P. aeruginosa quorum regulons led to an unexpected and interesting finding. We examined a LasR, RhlR, QscR signal receptor triple mutant to which we added 3OC12-HSL. Without the signal receptors we expected no response to 3OC12-HSL. To our surprise two operons, antABC and catBCA were activated by the signal. These operons encode enzymes for the degradation of anthranilate to TCA (tricarboxylic acid) cycle intermediates. Anthranilate is alternatively used by P. aeruginosa for the synthesis of PQS (Pseudomonas quinolone signal). We found that relatively high, yet physiological, concentrations of the P. aeruginosa signal 3OC12-HSL are required for activation of antABC and catBCA. In comparison, slightly lower levels of certain acyl-HSLs made by other bacterial species activate antABC and catBCA. Although P. aeruginosa has multiple pathways for deriving anthranilate, the kynurenine pathway is the critical source of anthranilate for PQS production. Interestingly, we found that this pathway is also the source of anthranilate for energy metabolism via the antABC gene products. Ongoing experiments suggest that acyl-HSL regulation of the ant and cat operons may be achieved by modulation of levels of the inducer, anthranilate or AntR, the transcriptional activator of the ant operon. Our findings are particularly interesting for two reasons. I. As far as we know this would be the first example of post-transcriptional control by an acyl-HSL. II. This control operates at an intricately regulated metabolic branch point that determines whether anthranilate is utilized for energy production or PQS synthesis.


Multiple distinct mechanisms integrate to control transcription from the unusual s70-Pr promoter for the aromatic-sensor DmpRTeresa del Peso Santos1, Peter Togneri1, Linda Johansson1, Lisandro Bernardo1 and Victoria Shingler1

1Molecular Biology, Umeå University, 901 87, Umeå, Sweden

DmpR is obligatory for transcription from the s54-dependent Po promoter, which controls transcription of an operon encoding a suit of enzymes for catabolism of (methyl)phenols by Pseudomonas putida. The aromatic-sensor DmpR takes up its active transcriptional promoting conformation upon binding phenolic effectors. The s54-Po promoter and the s70-dependent Pr promoter that controls transcription of dmpR drive divergent but non-overlapping transcription from an intergenic region. Pr is an extended -10 s70-promoter that requires the region from -38 to +1 relative to the transcriptional start for activity. However, this promoter intrinsically functions poorly, and its output is intricately regulated (1). Two mechanisms stimulate transcription from Pr: I) occupancy and activity of the

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divergent s54-Po promoter, and II) the co-action of the alarmone ppGpp and its co-factor DksA, which act together through binding RNA polymerase to modulate transcription from susceptible promoters. These two mechanisms place s70-Pr under control of signals that elicit s54-Po activity to create a feed forward loop, and effectively couple efficient transcription from Pr to low energy conditions (1). In addition, the dmpR mRNA has an extensive 5' untranslated leader region (5'-UTR); the presence of the DNA encoding the 5'-UTR results in ~5-fold decreased output from Pr by some unknown mechanism. The transcriptional start site from Pr is located unusually far (29 bp) from the promoter. Here, as a strategy to probe steps that limited output from Pr, and thus represent potential targets for the regulation of Pr activity, we sought to identify promoter-up mutations. Random and scanning mutagenesis of the 29 bp region between Pr and the +1 start identified three substitutions that give rise to high levels of Pr activity in vivo and in vitro. The most potent of these substitutions results in ~100-fold higher transcription in intact cells. Our in vitro and in vivo analysis of the effects of these substitutions on different steps of transcriptional initiation (RNA polymerase binding, open-complex formation and stability), together with the results of manipulations of the 5'-UTR encoding region, will be presented alongside their mechanistic implications.1. Johansson et al. (2008). s54-RNA polymerase controls s70-dependent transcription from a non-overlapping divergent promoter. Mol. Microbiol. 70:709 s = sigma


Population density and growth rate influence quorum sensing signalling molecule synthesis in Pseudomonas aeruginosa PAO1Jean-Frederic Dubern1, Catherine Ortori 2, Nigel Halliday 3, Kim Hardie 3, Dave Barrett 2, Miguel Camara3 and Paul Williams 3

1Centre for Biomolecular Sciences, 2Centre for Analytical Biosciences, School of Pharmacy, 3Centre for Biomolecular Sciences, School of Molecular Medical Sciences, Nottingham, UK

The opportunistic pathogen Pseudomonas aeruginosa produces a number of quorum sensing signal molecules (QSSM) including two major N-acyl-L-homoserine lactones (AHLs): (N-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), synthesised via LasI and RhlI respectively. In addition it also makes over 50 different types of 4-quinolone molecules (AQs of which 2-heptyl-3-hydroxyquinolone (PQS) and 2-heptyl-4-quinolone (HHQ) function as QS signals . The synthesis and bioactivity of AQs is controlled via the las and rhl quorum sensing systems. QSSMs are derived from the intermediates of relatively few biochemical pathways - in particular, fatty acid biosynthesis and the activated methyl cycle (AMC). Acyl-ACPs are required for the biosynthesis of both AHLs and 4-Qs and the AMC provides precursors for AHL (i.e. S-adenosylmethionine (SAM)) biosynthesis. In addition, AQ biosynthesis requires anthranilate.Despite knowledge that production of QSSMs is directly linked to the availability of starting molecules generated through the assimilation of nutrients by the cell, the metabolic control of QSSM synthesis is still poorly understood, particularly as a function of growth environment, growth rate and population density. The aim of this study was to demonstrate that by using QSSMs derived from central precursors such as SAM, bacteria control their collective behaviour at the metabolic as well as genetic level. To explore the impact of the AMC on QS as a function of growth environment, P. aeruginosa PAO1 was cultured in a chemostat using chemically defined simple salts minimal medium limited for specific nutrients under controlled temperature, oxygenation, and growth rate. Using different growth limiting substrates, the impact of growth rate and cell population density on bacterial AMC metabolite pools and QSSM synthesis was investigated. In this study AHLs and AQs, as well as metabolites of the AMC, de novo synthesis of sulphur amino acids, and related pathways (SAM, SAH, methionine, homocysteine, homoserine), were profiled and quantified using LC-MS-MS during steady state growth The same samples were also used for transcriptome experiments and virulence determinant assays to obtain holistic insights into the impact of QS on P, aeruginosa physiology as a function of environment, growth rate and population density.


Modulation of the c-di-GMP turnover by two new integral sensor proteins in the opportunistic pathogen Pseudomonas aeruginosaMichael Entian1 and Nicole Frankenberg-Dinkel1

1Ruhr-University Bochum, Physiology of Microorganisms, Germany

In order to cope with different environmental conditions bacteria have evolved a variety of different regulatory systems. One of these is a two component system, which are typically composed of a sensor histidine kinase and a corresponding response regulator. In addition other regulatory elements can be found which sense environmental stimuli and convert them into internal signals. A key player in such a system is the novel second messenger cyclic (c)-di-GMP, which is involved in the regulation of different cellular functions like motility or biofilm formation. Two distinct proteins are involved in the turnover of c-di-GMP: GGDEF-domains harbour diguanylate cyclase activity and build up c-di-GMP from two molecules of GTP, while EAL- domains act as phosphodiesterases and break down c-di-GMP into two molecules of pGpG. Like other bacteria, Pseudomonas aeruginosa has an abundance of proteins with a GGDEF and/or an EAL domain. These domains are often combined with signal input domains reflecting the ability to react to different environmental conditions. One novel sensor domain which is connected with GGDEF and EAL domains is the so called MHYT domain. Here we present a biochemically approach to analyse the function of two novel membrane sensor proteins in Pseudomonas aeruginosa. The gene products of PA1727 and PA3311 consist of an N-terminal membrane sensor domain MHYT and a C-terminal cytoplasmic GGDEF- and EAL-domain. Different variants of both proteins were constructed and tested for their catalytic activity. In vitro enzymatic activity assays showed that PA1727 has diguanylate cyclase activity. Similar activity was also shown for PA3311 but with a much slower turnover time, which is most likely due to an imperfect GGDEF motif. To gain more detailed insights into substrate binding and conversion, residues in the active sites were mutagenized. Conversion of GGDEF to AGDEF in PA1727 led to decreased diguanylate cyclase activity, whereas substitution of AGDEF to GGDEF in PA3311 increased the activity. Although the domain structure is similar in both proteins our data suggest that the two proteins might have opposing activities. Further experiments to test this hypothesis will be presented.


Transcriptomic analysis of 2-alkyl-4-quinolone signalling in Pseudomonas aeruginosa PAO1Matthew P Fletcher1, Giordano Rampioni1, Victoria Wright1, Steve Diggle1, Miguel Camara1 and Paul Williams1


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Quorum sensing (QS) is a bacterial intercellular communication system which depends on the production and sensing of small diffusible signal molecules. In Pseudomonas aeruginosa, QS constitutes a sophisticated global regulatory network controlling hundreds of genes that employ N-acylhomoserine lactone (AHL) and 2-alkyl-4-quinolones (AQ) signal molecules. The major P. aeruginosa AQ signal molecules are 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ). HHQ is synthesized via the products of first four genes of the pqsABCDE operon, and is converted to PQS by the mono-oxygenase, PqsH. An additional mono-oxygenase (PqsL) is required for synthesis of the N-oxide derivatives of AQs which are potent cytochrome inhibitors but their contribution to QS, if any, is not known. Both HHQ and PQS bind and activate the transcriptional regulator PqsR that increases transcription of the pqsABCDE operon, generating a positive feedback loop. The pqsE gene codes for a predicted metallo-beta-lactamase that is not required for HHQ or PQS biosynthesis or action, but induces the AQ-independent expression of virulence factors including pyocyanin and lectin. High density oligonucleotide microarrays comparing the transcriptomes of P. aeruginosa PAO1 with isogenic pqsA, pqsH, pqsL and pqsE mutants respectively have been used to clarify the physiological roles of PQS, HHQ, 2-heptyl-4-quinolone N-oxide (HHQNO). Preliminary investigations have revealed both specific and overlapping regulons associated with each AQ, whereas analysis of the pqsE regulon has highlighted a role for PqsE in controlling both AQ-dependent and independent genes. These include transcriptional regulators, enzymes involved in anaerobic respiration, iron acquisition, biofilm formation and virulence determinants.


Characterization of the Pseudomonas aeruginosa cAMP phosphodiesterase CpdA and its role in virulence factor regulationErin L. Fuchs1 and Dr. Matthew C. Wolfgang1

1University of North Carolina-Chapel Hill, USA

In P. aeruginosa, the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) plays an essential role in virulence regulation. cAMP is an allosteric regulator of the transcription factor Vfr, which controls expression of ~200 genes involved in virulence. While adenylate cyclases involved in cAMP synthesis have been identified, enzymes involved in cAMP degradation have not been characterized for P. aeruginosa. Based on homology to previously characterized enzymes, the PA4969 gene product of P. aeruginosa (CpdA) is predicted to be a Class III cAMP phosphodiesterase. In this study, the role of CpdA in P. aeruginosa cAMP homeostasis was determined.We have demonstrated that purified CpdA has cAMP phosphodiesterase activity in vitro and that this activity is stimulated by iron. Furthermore, CpdA activity was eliminated upon alanine substitution of conserved residues predicted to coordinate iron within the active site. These results are consistent with the notion that P. aeruginosa CpdA is a metallophosphodiesterase similar other Class III cAMP phosphodiesterases.To determine the role of CpdA in controlling intracellular cAMP levels, we constructed a mutant lacking the gene encoding CpdA. Direct cAMP measurements revealed that the cpdA mutant contained ~4 fold higher cAMP levels than the wild type strain. cAMP levels were also elevated in cpdA strains lacking the adenylate cyclase CyaB. Since CyaB synthesizes the majority of cellular cAMP, these results indicate that CpdA and CyaB may be the major players in balancing cAMP levels for appropriate virulence factor expression. Indeed, cAMP-Vfr dependent expression of the virulence factors ExoS, ToxA, and protease IV was elevated in the cpdA strain compared to wild type. However, preliminary studies suggest that cpdA is attenuated in a mouse model of acute pneumonia. Taken together, these data suggest that CpdA optimizes the level of intracellular cAMP required for P. aeruginosa infection.We have also characterized a slow growth phenotype associated with the loss of CpdA. The defect was only observed in rich media and was not due to elevated cAMP levels since cpdA mutants lacking the ability to synthesize cAMP also exhibited slow growth. Wild type growth could be restored following complementation with either P. aeruginosa cpdA or the homologous E. coli cpdA gene, indicating that Class III cAMP phosphodiesterases may have a previously unrecognized function beyond their role in cAMP metabolism.


A eukaryotic-type signaling system of Pseudomonas aeruginosa contributes to stress resistance and virulenceJana Goldova1 and Pavel Branny2

1Institute of Microbiology, 2Institute of Microbiology, Division of Cell and Molecular Microbiology, Videnska 1083, Prague 4, 142 20, Czech Republic

Eukaryotic-type serine/threonine protein kinases and phosphatases are widespread in many bacteria, although little is known regarding the cellular processes they control.The genome of P. aeruginosa includes at least three genes encoding putative Ser/Thr protein kinases, one of which, ppkA has been implicated in P. aeruginosa virulence. In this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA of P. aeruginosa.Double mutant strain ΔpppA-ppkA prepared in PAO1 background was viable and had a significantly reduced growth rate compared to that of the parent strain. Deletion also resulted in delayed production of virulence factors such as rhamnolipids, elastases or pigments pyocyanin and pyoverdine.To determine whether ΔpppA-ppkA strain has an altered sensitivity to different stress conditions, we examined its ability to survive H2O2-induced oxidative stress as well as its ability to cope with high osmolarity in vitro. Mutant strain showed a decreased resistance to oxidative stress and increased resistance to osmotic stress indicating that these signaling proteins affects the functions involved in stress resistance. Consistently, when intracellular survival of P. aeruginosa strains in macrophage-mediated bactericidal assay was examined, pppA-ppkA mutant showed decreased survival rate when compared to the WT. Furthermore, deletion of both genes compromises P. aeruginosa in the plant virulence model.To begin to address the genetic basis of the phenotype, we performed global transcriptome analysis of the pppA-ppkA mutant and the PAO1 parent grown in minimal medium. Several functional gene categories have been identified that could account for a reduced stress response and attenuated survival. Affected genes can be clustered into four regulons: i) response regulator PrpB regulon; ii) stationary phase sigma factor RpoS regulon; iii) PQS (Pseudomonas quinolone signal) biosynthetic pathway; iv) oxidative stress-responsive genes.Comparison of the whole-genome expression profiles of the wild type and mutant strains grown under the conditions of oxidative stress identified several gene categories previously showed to be associated with the oxidative stress and confirmed decreased adaptability of the mutant to the stress.In summary, our studies show that effect of the pppA-ppkA mutation is broadly pleiotropic and that kinase-phosphatase couple functions as a global regulator of gene expression in P. aeruginosa.

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The RND-type multidrug efflux system MexAB-OprM of Pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell to cell communicationNaomasa Gotoh1, Shu Minagawa1, Hiroyuki Inami1, Tomohisa Kato1, Shinji Sawada1, Shinichi Miyairi2 and Jun Okuda1

1Kyoto Pharmaceutical University, 2College of Pharmacy, Nihon University, Japan

Gram-negative bacteria use a variety of self-produced chemical signals (autoinducers) such as acylated homoserine lactones as a language for cell-to-cell communication, which is called “Quorum Sensing (QS)”, within and between bacterial species. This signal transduction system plays a significant role both in the pathogenesis of infectious disease and in beneficial symbiosis. However, QS requires to a cost of producing the autoinducers or of performing the cooperative behavior that is coordinated by QS. The human opportunistic gram-negative bacterium Pseudomonas aeruginosa strongly responds to 3-oxo-C12-HSL, rather than other short or long chained 3-oxo-acyl-HSLs. However, crystal and in silico analyses of LasR, a receptor for 3-oxo-acyl-HSLs, indicated that a length of acyl chain is not a major factor for binding between LasR and acyl-HSLs. These findings led to the hypothesis that an RND-type xenobiotics efflux pump MexAB-OprM of P. aeruginosa might function to selection of acyl-HSLs including P. aeruginosa cognate autoinducer, 3-oxo-C12-HSL.lasB promoter-gfp fusion reporter assay and LasB (elastase) assay, based on the finding that expression of lasB is regulated by the LasR-3-oxo-acyl-HSLs complex, resulted that loss of the MexAB-OprM due to deletion of mexB causes increases of QS responses, due to binding of 3-oxo-acyl-HSLs to LasR, to exogeneous HSLs substituted by 3-oxo-acyl-groups of carbon numbers of 9 and 10, rather than the native 12. Thus, we concluded that the MexB component of MexAB-OprM pump functions to selection of 3-oxo-acyl-HSLs by its extrusion ability, supporting by an in vivo experiment that a MexAB-OprM deletion mutant showed a strong QS response caused by co-incubation with Vibrio anguillarum producing 3-oxo-C10-HSL. Furthermore, contribution to decreases of a cost of performing the cooperative behaviour by selection of 3-oxo-acyl-HSLs by the MexAB-OprM pump suggested by a long time incubation experiment that QS-defective mutants were more resistant to cell lysis and death than wild type strain.


Similarities between the 2-alkyl-4-quinolone systems of Pseudomonas aeruginosa and Burkholderia species: functions and signalling interactionsMarie-Christine Groleau1, Jean-Philippe Dumais1, Ludovic Vial1, François Lépine1 and Eric Déziel1

1INRS-Institut Armand-Frappier, Laval, Canada

The pqsABCDE operon in Pseudomonas aeruginosa is centrally involved in the biosynthesis of a large variety of 4-hydroxy-2-alkylquinolines (HAQs), which include the Pseudomonas Quinolone Signal (PQS) and the precursor signal 4-hydroxy-2-heptylquinoline (HHQ). Besides their role in quorum sensing (QS), HAQs (also known as 2-alkyl-4-quinolones) have been implicated as antimicrobials, as iron chelators, and as mediators of membrane vesicles (MVs) formation. Although it previously seemed that HAQs were a feature of P. aeruginosa, the presence of a homologous pqs operon in Burkholderia pseudomallei, B. thailandensis and B. ambifaria was recently reported, and HAQs similar to those produced by P. aeruginosa were detected in cultures. Burkholderia cepacia complex (such as B. ambifaria) species comprise opportunistic human pathogens that cause severe respiratory infections in people suffering from cystic fibrosis. B. pseudomallei causes potentially fatal septicemic infection of animals and humans. B. thailandensis is very closely related to B. pseudomallei but much less virulent. Interestingly, in these species, the pqs operon contains two additional genes downstream of the pqsE homologue, one we found coding for a methyltransferase. Accordingly, in contrast with those of P. aeruginosa, Burkholderia HAQs are methylated (at the 3 position), hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). Consequently, the biosynthesis operon was named hmqABCDEFG. We are investigating the functions and properties of HMAQs. In P. aeruginosa, the production of HAQs is autoregulated, as both PQS and HHQ act as co-inducers of MvfR/PqsR, the transcriptional regulator controlling pqsABCDE. In contrast, no MvfR-type regulator is found in hmqABCDEFG-carrying Burkholderia species, and HMAQs do not stimulate their own production. Instead, we found that, in B. ambifaria, the LuxIR-type QS regulatory system (BafIR) directs the transcription of hmqABCDEFG via the C8-homoserine lactone (HSL) autoinducer. Moreover, unlike in P. aeruginosa, the 2-alkyl-4-quinolone system of Burkholderia affects the LuxIR-type QS system: mutants defective in the synthesis of HMAQs overproduce C8-HSL which, consequently, influences QS-regulated phenotypes. Studies on the interactions between the P. aeruginosa HAQs and Burkholderia HMAQs systems revealed that HMAQs can activate transcription of the heterologous pqsABCDE operon but that the methyl group critically impacts this interaction.


GidA controls quorum sensing in Pseudomonas aeruginosa Rashmi Gupta1, Timothy Gobble2 and Martin Schuster3

1Oregon State University, 2School of Medicine, Loma Linda University, Loma Linda, California 92350, 3Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis OR, 97331, USA

The opportunistic human pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum sensing (QS) systems, LasRI and RhlRI, to regulate expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of co- and superregulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a non-redundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS. We initially screened the entire library for loss-of-function in one QS-controlled trait, and then re-screened candidates for defects in independent QS-phenotypes to exclude mutants defective in functions other that QS. We re-identified several known regulatory genes, but only identified two novel genes, gidA and

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pcnB that affected multiple QS-controlled traits. We characterized gidA further. This gene is predicted to encode an FAD-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as pyocyanin and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on acyl-HSL production. Complementation of a gidA mutant with rhlR partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA controls QS gene expression posttranscriptionally via RhlR-dependent and independent pathways. The selectivity of GidA towards rhl-dependent QS gene expression suggests a regulatory function other than global tRNA modification in P. aeruginosa.


The Pho regulon influences biofilm formation and type three secretion in Pseudomonas aeruginosaAhmed Mahmoud Haddad1, Vanessa Jensen1 and Susanne Häussler1

1Zell- und Immunbiologie, Helmholtz Zentrum für Infektionsbiologie, Braunschweig, Germany

Research into the molecular mechanisms of the switch from highly motile to biofilm forming Pseudomonas fluorescens bacteria recently uncovered a role of inorganic phosphate as an important environmental regulatory factor to control c-di-GMP levels in the cell. In this study we present evidence that in the opportunistic pathogen P. aeruginosa the Pho regulon inhibits biofilm formation and is required for the repression of the type three secretion system. We furthermore identified an EAL domain protein, referred to as RapA, as a down-stream effector of the Pho regulon, which at least partially mediated the observed inhibition. Interestingly, inhibition of the P. aeruginosa PA14 virulence phenotype was Pho regulon dependent, but independent of the availability of inorganic phosphate, suggesting that the expression of a basal level of PhoB even under high phosphate is sufficient for the repression of biofilm formation and type three secretion. These results clearly show that the Pho regulon contributes to the expression of the virulence phenotype in P. aeruginosa and add even more complexity to the regulation of the bacterial behaviour by environmental cues.


A ChIP-chip approach uncovers co-operative target binding of Pseudomonas aeruginosa RpoS and the quorum sensing regulators LasR and RhlRSusanne Häussler1, Vanessa Jensen1, Juliane Schmidt1, Sebastian Bruchmann1, Andreas Dötsch1, Giuliano Degrassi2, Robert Geffers1

and Vittorio Venturi2

1Cell- and Immune Biology, Helmholtz Centre for Infection Research, Germany, 2International Centre for Genetic Engineering, Trieste, Italy

In Pseudomonas aeruginosa the stationary phase alternative sigma factor, RpoS, governs a complex regulon, which can channel cellular responses to several environmental stresses through the redirection of transcription initiation. Since RpoS also controls quorum sensing gene expression in P. aeruginosa, the aim of this study was to elucidate the interdependencies of the RpoS and the quorum sensing regulatory networks. We searched for direct RpoS target promoter sequences by performing chromatin immunoprecipitation coupled to a P. aeruginosa genome microarray (ChIP-chip). Using this genome-wide approach, we identified 496 RpoS binding sites with high reproducibility. Categorization of the target sequences revealed that a large subset harbours a RpoS binding sequence motif and/or the respective down-stream genes have previously been shown to be transcriptionally regulated by RpoS. However, we also identified a subset of targets that lacked a common RpoS motif and only bound RpoS in the presence of LasR or both LasR and RhlR. Since a LexA-based protein interaction system revealed that LasR and RpoS directly interact, our findings do not only support previous models of the complex relationship between RpoS and quorum sensing, but also opens a window for the elucidation of transcriptional regulatory mechanisms of RpoS in other bacterial pathogens.


Pseudomonas aeruginosa cyclic di-GMP signalling: Insight into function of a novel membrane anchored GGDEF/EAL sensorSabrina Heine1, Michael Entian1 and Nicole Frankenberg-Dinkel1

1Ruhr-University Bochum, Physiology of Microorganisms, Germany

Cyclic di-GMP is a novel second messenger in bacteria that is involved in the regulation of multiple cellular functions including motility, adhesion, biofilm development and expression of virulence genes. The overall turnover of cyclic di-GMP in bacteria is affected by synthesis and degradation, whereas GGDEF protein domains are involved in the conversion of two molecules of GTP to cyclic di-GMP and EAL domains in cyclic di-GMP breakdown to pGpG. In bacteria, numerous proteins involved in cyclic di-GMP conversion are found. These proteins show a high diversity in protein domain organisation containing: (I) only GGDEF domains; (II) only EAL domains as well as (III) both GGDEF and EAL domains. Additionally, these protein domains (I)-(III) are also combined with diverse signal input domains suggesting an association with signal transduction in bacteria. Pseudomonas aeruginosa features about 40 genes encoding proteins possibly involved in cyclic di-GMP signalling. On the basis of amino acid sequence analyses two GGDEF-EAL proteins of P. aeruginosa (PA1727 and PA3311) could be identified linked with the newly-discovered membrane sensor domain, MHYT. This domain consists of several transmembrane segments and is postulated to play a role in sensing diatomic gases. In case of the three-domain proteins PA1727 and PA3311 signals could be sensed by the MHYT domain and then converted by either the GGDEF domain or EAL domain into an intercellular cyclic di-GMP signal. While PA1727 (MucR) was shown to be involved in regulation of alginate production [1], the function of PA3311 remains unknown.To investigate the function of PA3311 a combination of genetic, transcriptional and phenotypic analyses was employed. Colonies of a PA3311 transposon mutant showed an increased production of mucoid substances and a decreased viability under anaerobic conditions. As O2 and NO are know to impact alginate production these results support a role of PA3311 in alginate production as shown for MucR (PA1727). However, in contrast to MucR the GGDEF domain from PA3311 exhibits an imperfect active site with the amino acid

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sequence AGDEF pointing to an inactive GGDEF domain. Motility assays with the transposon mutant support this assumption as a reduced swarming behaviour was observed pointing towards an active EAL domain but an inactive GGDEF domain.[1] Hay, I. D., Remminghorst, U. & Rehm, B. H. (2009). MucR, a novel membrane associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol 75, 1110-1120.


Structural and functional characterization of the Pseudomonas Quinolone Signal (PQS) Response Protein PqsE Vanessa Jensen1, Shen Yu2, Janine Seeliger2, Ingo Feldmann3, Susanne Häussler1 and Wulf Blankenfeldt2

1Zell- und Immunbiologie, Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany, 2Strukturelle Enzymologie, Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany, Institute for Analytical Sciences, Dortmund, Germany

In bacteria, the transcription of most virulence genes is controlled by a cell-density dependent process known as 'quorum sensing' (QS). QS relies on small diffusible signalling molecules that cross the bacterial cell wall and activate target transcription factors after a threshold concentration has been reached. Besides two hierarchical QS circuits based on N-acyl-homoserine lactones, the human opportunistic pathogen Pseudomonas aeruginosa integrates a signaling system that depends on 2-heptyl-3-hydroxy-4-quinolone, termed 'Pseudomonas Quinolone Signal' (PQS). PQS is produced from genes encoded by the pqs-operon, which in addition to the biosynthetic enzymes PqsA-D contains a fifth gene pqsE that is not required for production of PQS but whose disruption leads to loss of signal transduction in several but not all pqs-operon-dependent processes. PqsE was hence termed 'PQS response protein', but its exact mechanism of action is unknown. We have determined the crystal structure of PqsE and show that it possesses a metallo-beta-lactamase fold with two iron atoms bound to the active center. A copurified ligand was assigned as benzoate and may indicate that PqsE exerts its regulatory effect by converting a chorismate-derived molecule. Further, PqsE was found to slowly hydrolyze bis-pNPP, indicative of phosphodiesterase activity. Low activity was also observed towards single and double-stranded DNA as well as towards mRNA. Based on these results several hypotheses about the physiological roles of this enzyme are discussed.



Contrasting roles of different signal-transducing proteins in Pseudomonas aeruginosaIain L Lamont1 and Karla Mettrick1

1University of Otago, New Zealand

Pseudomonas aeruginosa can take up a wide variety of iron-chelating compounds (siderophores) in order to acquire iron from the extracellular environment. Uptake occurs via cell surface receptors that are specific to different ferrisiderophores and enable their uptake across the outer membrane, with the iron being released for incorporation into the cellular iron pool. Production of a number of ferrisiderophore receptors is induced by the presence of the corresponding ferrisiderophore. Synthesis of the receptors FpvA, FiuA and FoxA for uptake of the siderophores pyoverdine, ferrichrome and desferrioxamine respectively is directed by specific sigma factors (FpvI, FiuI or FoxI). The activity of each sigma factor is controlled by a corresponding signal-transducing protein (FpvR, FiuR or FoxR). These proteins span the cytoplasmic membrane, interacting with the receptor proteins to control the activities of the sigma factors in response to the presence (active sigmas) or absence (inactive sigmas) of the relevant ferrisiderophore.Here we show that the predicted cytoplasmic portions of the signal-transducing proteins are sufficient to control sigma factor activity but that the systems work in different ways. Deletion of the entire fpvR gene resulted in the FpvI sigma factor being active and the target fpvA gene being expressed even when pyoverdine was not present showing that FpvR is an inhibitor of FpvI activity. Deletion of the part of fpvR corresponding to the periplasmic part of the protein resulted in inhibition of FpvI action independent of the presence of pyoverdine. In contrast, deletion of the entire foxR and fiuR genes resulted in greatly reduced sigma factor activity even when desferrioxamine and ferrichrome were present, showing that FoxR and FiuR are required for sigma factor activity. Truncated versions of foxR and fiuR that encoded proteins with part of the periplasmic domain missing resulted in the corresponding sigma factors being active whether or not siderophore was present. These data show that in all three cases the periplasmic portion of the signal transducing protein is required for controlling the activity of the cytoplasmic portion in response to the presence or absence of siderophore. However, the cytoplasmic portions work in different ways to control sigma factor activity.


The luxR family transcriptional regulator RfiA play an indispensable role in Pseudomonas corrugata virulence on tomatoGrazia Licciardello1, Iris Bertani2, Laura Steindler2, Patrizia Bella1, Vittorio Venturi2 and Vittoria Catara1

1Department of Phytosanitary Science and Technology, University of Catania, Italy, 2Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano, Trieste, Italy

Pseudomonas corrugata is a ubiquitous bacterium first described as causal agent of "pith necrosis" on tomato and isolated in all tomato-growing areas of the world. Considerable research activity has established that it can act as PGPR and as biological control agent (BCA) above all against soilborne pathogens (Catara et al., 2007). Up to now, molecular studies on P. corrugata are limited only to a few aspects. For example, an important role is attributed to phytotoxic and antimicrobial cationic lipodepsipeptides (corpetin A and corpeptin B). In a previous study the pcoI/pcoR N-acyl homoserine lactone (AHL) quorum sensing (QS) system has been isolated and as assessed by mutant derivatives, QS has an important role in virulence and in vitro antagonistic activity (Licciardello et al., 2007).

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Here we describe a gene, rfiA, encoding for a transcriptional regulator belonging to the LuxR family located downstream the pcoI gene. RT-PCR analysis revealed that rfiA is directly linked to QS by co-transcription with pcoI. In the downstream region of pcoI-rfiA, a RND (Resistance Nodulation System) transport system which is directly regulated by RfiA was identified.We found that pcoI, pcoR and rfiA knock-out mutant cultural filtrates are unable to inhibit the growth of Rhodotorula pilimanae and Bacillus megaterium microrganisms, indicators used to detect the LDPs production. Moreover, time course monitoring revealed that both AHLs and LDPs are produced at high bacterial population cell densities.The importance of RfiA in the development of disease symptoms in tomato plants was demonstrated by the absence of necrosis within the stem pith tissues inoculated with its mutant as compared to those inoculated with the wild-type strain.Our data strongly suggest that P. corrugata QS regulates LPDs production through the transcriptional activator RfiA which then regulates the transcription of genes responsible of toxin production and secretion. Catara, V. 2007. Pseudomonas corrugata: plant pathogen and/or biological resource? Mol. Plant Pathol. 8:233-244.Licciardello, G., Bertani, I., Steindler, L., Bella, P., Venturi, V., and Catara, V. 2007. Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; role in tomato pathogenicity. FEMS Microbiol. Ecol. 61:222-234


Inhibition of quorum sensing by natural plant productsKalai Mathee1, Allison Adonizio1, Sixto Leal1, Kok-Fai Kong1, Jessica Dawlaty2, Natalie Maricic1, Bharathi Ramalingam1, Christian Villaseca1, Horacio Priestap1, John Makemson1, J. Martin Quirke1, Frederick Ausubel2, Jon Clardy2 and Lisa Schneper1

1Florida International University, Miami, USA, 2Harvard Medical School, Boston, USA

The elaboration of a myriad of virulence factors coupled with an innate ability to counteract antibiotics and immune response has made Pseudomonas aeruginosa the number-one killer of cystic fibrosis patients. Virulence factor expression in P. aeruginosa is under the control of quorum sensing (QS), a method by which the bacteria monitor population density and mount responses that are important for establishing an infection. Thus, the identification of compounds that interrupt quorum sensing without inducing selection for antibiotic resistance is of medical importance. Using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens, extracts from six South Florida plants, Conocarpus erectus, Chamaecyce hperififolia, Callistemon viminalis, Bucida burceras, Tetrazygia bicolor, and Quercus virginiana, were found to inhibit QS. Experiments using the aforementioned assays as well as Vibrio harveyi bioluminescence assay have identified anti-QS activity in extracts from Panax ginseng CA Meyer. Three of the South Florida plants, C. erectus, B. buceras, and C. viminalis, differentially affected virulence factor expression and biofilm formation in P. aeruginosa. These extracts were also assayed for their ability to reduce killing of the nematode Caenorhabditis elegans by P. aeruginosa PA14 and PAO1 virulence. Mortality from gut infection and death from toxin production was reduced in the nematodes treated with the extracts without host toxicity. Two compounds responsible for anti-QS activity were purified from an aqueous extract of C. erectus. The stereoisomeric C-glycosidic ellagitannins, vescalagin and castalagin, were isolated using bioassay-guided fractionation and HPLC. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to significantly decrease AHL signal production, QS gene expression, and virulence factor production in P. aeruginosa. This is the first report of vescalagin and castalagin being isolated from C. erectus, and also the first report of their activity on the QS system of P. aeruginosa. Together, this data suggests that these plant compounds have potential for further development as anti-infective therapeutic agents.


Biosynthetic production of alkylquinolonesHeiko Niewerth1, Catherine Ortori2, Siri Ram Chhabra3, Paul Williams3 and Susanne Fetzner1

1Institute of Molecular Microbiology and Biotechnology, WWU Münster, Germany, 2School of Pharmacy, University of Nottingham, UK, 3Centre for Biomolecular Sciences, University of Nottingham, UK

2-Alkyl-4(1H)-quinolones (AQs) were identified already in the 1940s as antimicrobial secondary metabolites of fluorescent pseudomonads. 2-Heptyl-3-hydroxy-4(1H)-quinolone and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) were later found to act as signalling molecules in the quorum sensing system of Pseudomonas aeruginosa [1, 2]. 2-Heptyl-3-hydroxy-4(1H)-quinolone, referred to as the Pseudomonas quinolone signal (PQS), regulates a diverse set of target genes, including genes coding for key virulence factors. Biosynthesis of HHQ via condensation of anthranilate and a beta-keto fatty acid is directed by the pqsABCD genes of P. aeruginosa; PqsH catalyzes the hydroxylation of HHQ to form PQS [3]. Since the availability of AQs is a prerequisite to further explore their significance in P. aeruginosa and other bacteria, we aimed at developing a system for their biosynthetic production. Pseudomonas putida KT2440 [pBBR1-pqsABCD] when grown in minimal medium supplemented with anthranilate and octanoate produces a series of AQ metabolites, with HHQ as the predominant AQ. About 30% and 70% of the HHQ was localized in the culture supernatant and the cell fraction, respectively. Analysis of the methanolic extract of biomass by HPLC and mass spectrometry indicated the presence of other AQs besides HHQ, with either a C9, a C11 or a C13 alkyl chain, or a monounsaturated C7/ C9/ C11 side chain, and traces of the C7 N-oxide. The biomass was extracted with methanol, whereas AQs from culture supernatants were isolated with an absorber resin. After concentrating the methanolic fractions, generation of a supersaturated solution by addition of cold water resulted in precipitation of AQs. From 8.7 mg of HHQ produced by an 800 ml culture, about 4.3 mg and 0.6 mg of HHQ were isolated from the biomass and from culture supernatant, respectively, corresponding to a yield of 56%. Hydroxylation of HHQ to PQS was achieved by whole-cell biotransformation using P. putida KT2440 [pBBR1-pqsH]; PQS was extracted from the biomass with methanol. The preparation and purification by preparative HPLC of HHQ, PQS and other biosynthetic AQs will facilitate studies on the biological activities of individual compounds. [1] Pesci et al. (1999) Proc Natl Acad Sci USA 96:11229-11234. [2] Deziel et al. (2004) Proc Natl Acad Sci USA 101:1339-1344.[3] Dubern & Diggle (2008) Mol BioSyst 4:882-888.


Characterization of PA3271 - an unorthodox sensor kinase in Pseudomonas aeruginosaJoerg Overhage1, Caroline Zaoui1, Vanessa Jensen1, Tanja Becker1 and Susanne Häussler1

1Helmholtz Center for Infection Research, Braunschweig, Germany

Pseudomonas aeruginosa uses multiple cell-to-cell signal molecules to control numerous cellular functions and virulence. The 4-quinolones, like the Pseudomonas quinolone signal (PQS) and its 2-heptyl-4-hydroxyquinoline (HHQ) precursor molecule, constitute

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one type of cell-to-cell signal molecules synthesized by this bacterium. During a screen for mutants deficient in 4-quinolone signaling, we identified a two-component sensor kinase transposon mutant (PA3271) with defects in the response to 4-quinolones. Phenotypic and transcriptomic analysis of several independent P. aeruginosa PA3271 sensor kinase mutants demonstrated its involvement in the oxidative stress response and its regulatory impact on the expression of a variety of different genes including bacteriophage synthesis, carbon and energy metabolism and siderophore biosynthesis among others. Enzymatic analysis revealed that the activity of this sensor kinase is dependent on the redox state of the respiratory chain ubiquinones, since an oxidized form of a ubiquinone analog inhibited the autophosphorylation activity of the purified cytosolic portion of the sensor kinase. This implies that the sensor kinase might be active during conditions of oxygen limitations, which leads to the accumulation of reduced ubiquinones. Overall, our data suggest that PA3271 is part of the 4-quinolones multi-step signaling cascade and that it is involved in triggering an oxidative stress response.


Characterization of new mutants affected in cell-cell communication of Pseudomonas aeruginosa PAO1Isabel Pérez-Martínez1, Cornelia Reimmann1 and Dieter Haas1

1Department of Fundamental Microbiology, University of Lausanne, Switzerland

In dense populations of many bacterial genera, cells communicate with one another by emitting and sensing chemical signals; these phenomena are commonly known as Quorum Sensing (QS). In the opportunistic pathogen Pseudomonas aeruginosa, QS regulation is highly complex and involves three known signal molecules. An additional, as yet uncharacterized signal molecule activates the two-component system GacS/GacA, which regulates the expression of several virulence genes, by positively controlling the expression of two small noncoding RNAs, RsmY and RsmZ. These riboregulators antagonize the repressive action of the RNA binding protein RsmA, which binds to target mRNAs near the translation initiation sites. Furthermore, the activity of the GacS/GacA system is modulated by two sensor kinases, RetS and LadS, which affect GacA activity by antagonizing or stimulating GacS, respectively. The presence of this system in several pathogenic and non-pathogenic γ-proteobacteria suggests the importance of this signal transduction pathway.By monitoring the rsmY promoter of an rsmY-gfp fusion in a collection of P. aeruginosa PAO1 Tn5-insertion mutants, we found several novel genes that affect the GacS/GacA pathway. When used as a control, a gacA mutant carrying the same construct was non-fluorescent. About 100 non-fluorescent or hyperfluorescent candidate colonies were obtained and the corresponding transposon insertions were localized in each candidate. This screen identified retS as a gene whose inactivation enhances the expression of the rsmY gene, as expected. Fifteen mutants were further analyzed in liquid media by testing the expression of rsmY- and rsmZ-lacZ fusions. Among the candidate mutants having a dim phenotype, one cluster containing conserved hypothetical genes was of interest. Among the hyper-fluorescent mutants, we noted a fumA candidate, suggesting that Krebs cycle function may be important for the expression of the GacS/GacA pathway.


Quenching of PQS-dependent quorum sensing in Pseudomonas aeruginosa by 2-methyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase (Hod)Christian Pustelny1, Alexander Albers2, Katja Parschat2, Siri Ram Chhabra1, Miguel Camara1, Susanne Fetzner2 and Paul Williams1

1Centre for Biomolecular Sciences, University of Nottingham, Nottingham, U.K, 2Institute of Molecular Microbiology and Biotechnology, University of Muenster, Germany

2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum sensing signal molecule employed by Pseudomonas aeruginosa which regulates 2-alkyl-4(1H)-quinolone (AQ) biosynthesis and the expression of key virulence genes. The inactivation of AQ signalling by mutagenesis of key AQ biosynthetic genes attenuates pathogenicity in experimental infection models. Consequently, the quenching of AQ-signalling via enzyme-mediated inactivation of PQS is an attractive strategy for the control of P. aeruginosa. The structural similarity between 2-methyl-3-hydroxy-4(1H)-quinolone (MPQS), the natural substrate for the Arthrobacter nitroguajacolicus Ru61a dioxygenase, (Hod (2-methyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase)) and PQS prompted us to investigate whether Hod could cleave PQS and quench AQ-dependent signalling in P. aeruginosa. Hod catalyzed the conversion of PQS resulting in the release of CO and the formation of N-octanoylanthranilic acid indicating that PQS undergoes 2,4-dioxygenolytic ring cleavage. The activity of Hod towards 2-alkyl-3-hydroxy-4(1H)-quinolones was highly dependent on alkyl chain length with the catalytic efficiency for PQS some 2000-fold less than the methyl congener, MPQS. Addition of the purified Hod protein to P. aeruginosa PAO1 cultures reduced expression of the PQS biosynthetic gene, pqsA and the PQS-regulated virulence determinants lectin A and pyocyanin although the efficiency of Hod as a quorum quenching agent was reduced by Pseudomonas exoproteases. Nevertheless, these data indicate that enzyme-mediated AQ inactivation has potential as anti-virulence strategy against P. aeruginosa.


Role of the RsaL quorum sensing regulator in Pseudomonas aeruginosa pathogenic potentialGiordano Rampioni1, Elisabetta Zennaro1 and Livia Leoni1

1University Roma Tre, Dept. of Biology, Rome, Italy

In the opportunistic human pathogen Pseudomonas aeruginosa three interconnected quorum sensing (QS) regulatory systems control the expression of virulence factors and biofilm formation in response to cell-density. In previous studies we showed that the RsaL transcriptional repressor governs a homeostatic circuit that maintains the levels of the major QS signal molecule (3OC12-HSL) within physiological profitable limits. Moreover we demonstrated that RsaL can regulate the expression of 130 genes, independently of its effect on 3OC12-HSL production. In order to assess the role played by RsaL in P. aeruginosa pathogenic potential, here we have compared the expression of phenotypes related to virulence and biofilm formation in the rsaL mutant and in the P. aeruginosa parental strain. The rsaL mutant overproduces secreted virulence factors (pyocyanin, hydrogen cyanide, elastase, proteases, hemolisins) and is hypervirulent in the Galleria mellonella acute infection model, with respect to the wild-type. Moreover, the rsaL mutant discloses on one hand an increased surface motility (twitching and swarming) and on the other hand reduced biofilm formation, with respect to the

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wild-type. In the whole, the phenotype of the rsaL mutant indicates that RsaL could be important for the establishment of chronic infections, characterized by limited virulence factor production and biofilm formation.This work was supported by the Italian Cystic Fibrosis Research Foundation (grants FFC#10/2007 and FFC#8/2008).


Quinolone signalling modulates global post-transcriptional regulation of gene expression in Pseudomonas aeruginosa Karima M Righetti1, Marco Messina1, Christian Pustelny1, Sarah Anna Kuehne1, Stephan Heeb1, Paul Williams1 and Miguel Cámara1


Since the discovery of quorum sensing (QS) signalling mechanisms in bacteria there have been many studies which have shown the importance of this type of regulatory mechanism in the global transcriptional control of gene expression. However there has been no clear evidence to attribute to QS a key role in post-transcriptional regulation of terminal gene targets. The sequencing of bacterial genomes from numerous species has allowed the prediction of a large number of small, non-coding RNAs many of which have been experimentally detected although no biological role has been assigned. Those of known function are often involved in regulating target mRNA translation. Using the model organism Pseudomonas aeruginosa, in which QS systems have been extensively studied, we found that the Pseudomonas Quinolone Signal (PQS) positively regulates the expression of the gene coding for the small RNA RsmZ. RsmZ antagonises by titration the effects of the global post-transcriptional regulator RsmA, a small RNA-binding protein which targets specific mRNAs. We show that RsmA binds to the 5' untranslated transcribed region of the PQS regulated lecA mRNA that encodes the PA-I galactophilic lectin, repressing its translation. As a consequence PQS controls lecA translation rates via the induction of rsmZ and the subsequent antagonism of RsmA activity. Furthermore, a transcriptional lecA-lux fusion made at the +1 was induced by PQS but in an RsmA-independent manner. Taken together these results demonstrate that QS not only controls terminal target gene expression at the transcriptional level, but also at the translational level. As the family of Rsm global regulators and many small RNAs appear to be present in a wide variety of bacterial species this opens the possibility that post-transcriptional control of gene expression by quorum sensing is widespread.


Characterization of the terminal hydroxylase responsible for PQS biosynthesisJeffrey W Schertzer1 and Marvin Whiteley1

1University of Texas at Austin, USA

Bacteria exhibit many social activities that are coordinated by complex cell-cell signaling systems, referred to as quorum sensing (QS). Pseudomonas aeruginosa is a prevalent cause of opportunistic infections due to inherent antibiotic resistance and the production of a myriad of virulence factors. Interestingly, QS is required for P. aeruginosa virulence, and the development of QS inhibitors has been proposed as a novel therapeutic strategy. To fully realize the potential of this approach, a detailed understanding of QS mechanisms will be required.The Pseudomonas Quinolone Signal (PQS) QS system exists at the interface between QS and virulence factor trafficking. PQS not only induces expression of virulence factors, but also physically promotes the formation of the outer membrane vesicles (OMVs) into which PQS and many virulence factors are packaged and trafficked. Little is known about this important process except that it is stimulated by PQS but not its immediate precursor HHQ. That these molecules differ only in the presence or absence of a single hydroxyl group at the 3rd position suggests a level of specificity inherent in OMV promotion. Chemically targeting the production or delivery of PQS through the use of small-molecule inhibitors could therefore provide a novel and effective approach to combating P. aeruginosa infections.The PqsH protein was predicted to possess HHQ hydroxylase activity based on its indispensability for PQS production and OMV formation, and its similarity to flavin-dependent monooxygenases. We have cloned and purified PqsH from P. aeruginosa PA14 and demonstrated PQS biosynthesis using HHQ as substrate. Thin layer chromatography of the reaction products identified a spot that fluoresced blue under UV light and co-migrated with an authentic PQS standard. Despite poor solubility of the enzyme, substrates and products, we developed both kinetic and end-point assays for steady state kinetic analysis of PqsH. The enzyme showed substrate-dependent production of PQS with Km values for HHQ and NADH of 2.5 µM and 20 µM, respectively. PqsH was localized to the periphery of the cell using a translational fusion to the yellow fluorescent protein. This localization was consistent with the likely site of synthesis of the hydrophobic secreted product. This work has given early insight into the location and mechanism of PQS biosynthesis and sets the stage for development of small-molecule inhibitors of P. aeruginosa QS.


Isolation and identification of c-di-GMP binding proteins in Pseudomonas aeruginosaJuliane Schmidt1, Daniela Bertinetti2, Michael Morr3, Frank Schwede4, Josef Wissing3, Lothar Jänsch3 and Susanne Häussler3

1Helmholtz-Zentrum für Infektionsforschung, 2Institut für Biologie/ Abteilung Biochemie, Universität Kassel, Kassel, Germany, 3Zell- und Immunbiologie, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany, 4Research and Developement, BIOLOG Life Science Institute, Bremen, Germany

Twenty years after its discovery bis-(3'-5') cyclic di-GMP (c-di-GMP) has come to be recognized as a ubiquitous second messenger in bacteria. c-di-GMP regulates a number of cellular functions like motility, the expression of virulence factors and biofilm formation. The cellular c-di-GMP level is tightly regulated by controlled synthesis and degradation of c-di-GMP, mediated by diguanylate cyclases containing a conserved GGDEF domain and by phosphodiesterases containing a conserved EAL domain, respectively. GGDEF and EAL domains are often fused to transmembrane and/or signal input domains. Thereby, c-di-GMP links environmental stimuli to an

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adaptive answer of the bacterial cell. However, little is known about the molecular details of the c-di-GMP signalling network. So far, PilZ domains are the only cellular c-di-GMP receptors yet identified, but others likely exist.The aim of our work is the identification and characterization of c-di-GMP binding proteins in the opportunistic pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa PAO1 encodes 38 proteins containing a GGDEF and/or EAL domain and 8 proteins with a PilZ domain. By using c-di-GMP coupled sepharose we have isolated PAO1 c-di-GMP binding proteins which were analyzed by mass spectrometry. Seven putative candidates have been identified, among others one protein with a PilZ domain and two proteins, with a GGDEF and a GGDEF and EAL domain, respectively. We are currently analyzing the phenotypes of the corresponding mutants to elucidate the cellular function of these proteins. In addition, we over-express and isolate the identified proteins for further in vitro studies. By illuminating details on the c-di-GMP signalling pathway we aim for a better understanding of the role of c-di-GMP in virulence on one side and persistence on the other.


Molecular analysis of the Roc signalling pathways in Pseudomonas aeruginosaMelissa Yap Sivaneson1, Christophe Bordi2 and Alain Filloux3

1Imperial College London, 2Centre National de la Recherche Scientifique, LISM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, 3Centre for Molecular Microbiology and Infection, Imperial College London, South Kensington Campus, SW7 2AZ, UK

The opportunistic pathogen Pseudomonas aeruginosa, a ubiquitous Gram- negative bacterium, is responsible for diverse acute infections in human that could lead to the death of immunocompromised patients. Moreover, this micro organism is able to cause chronic infections in human, especially in cystic fibrosis patients. Chronic infections have been associated with the capacity of P. aeruginosa to form biofilm, an organized and differentiated microbial community embedded within an extracellular matrix. Biofilm formation is a highly temporally and spatially regulated developmental process, involving many different surface appendages and regulatory pathways. One of these pathways is the two component system, named Roc1 after its role in the regulation of cup genes, which encode components of the chaperone/usher pathway- a cell-envelope machinery leading to the assembly of thin fimbriae at the surface of the bacterium. cup genes have been shown to be involved in biofilm formation. The Roc1 system is also involved in the regulation of the Type III secretion system which is commonly associated with acute infections. Hence, the Roc1 system appears to be an important regulatory pathway involved in the control of P. aeruginosa virulence and pathogenicity. The Roc1 system is composed of an unorthodox histidine kinase sensor (RocS1) and two response regulators: RocA1 with a C-terminal DNA binding domain and RocR with a C-terminal EAL domain (EAL domain is involved in the degradation of the second messenger cyclic di-GMP). There are two gene clusters encoding components homologous to the Roc1 system. The Roc2 system is composed of an unorthodox histidine kinase sensor (RocS2) and a single response regulator with a C-terminal DNA-binding domain (RocA2), whereas Roc3 is narrowed to a single orphan hybrid sensor (RocS3).Our work has shown that crosstalk occurs between the Roc signalling pathways, but does not necessarily control the expression of the same set of genes. We demonstrated that RocS2, like RocS1, controls the expression of cup genes through the RocA1 response regulator, but not RocA2. This novel finding led us to the characterization of a specific regulon of the RocA2 response regulator by using transcriptomic analysis and transcriptional lacZ fusions.It is anticipated that further molecular dissection of these complex regulatory pathways will reveal the powerful fine-tuning possibilities that bacteria have developed to respond to minuscule environmental changes.


RsmN, a new atypical RsmA homologue in Pseudomonas aeruginosaLaura Tye1, Marco Messina1, Elizabeth Morris2, Jed Long2, Mark Searle2, Stephan Heeb1, Miguel Cámara1 and Paul Williams1

1Institute of Infection, Immunity and Inflammation, 2School of Chemistry, Nottingham, UK

The RsmA/CsrA family of global post-transcriptional regulators are small RNA-binding proteins involved with the regulation of a large variety of genes such as those involved in quorum sensing, virulence factor biosynthesis, motility and biofilm formation. They bind to target mRNAs and hence modulate their stability and translation rates, having a negative effect when the binding occurs near ribosome binding sites. Their pleiotropic effects are antagonised when small non-coding regulatory RNAs, having little primary sequence conservation but high affinity for RsmA, are transcribed and titrate the protein away from target mRNAs. The control of expression of target genes via this post-transcriptional regulatory network is mostly operated in Pseudomonas spp. by the GacS/GacA two component system.Among the multiple effects caused by an rsmA mutation in P. aeruginosa PAO1, a reduction of rhamnolipid production and of swarming motility has been observed. The mechanism by which RsmA operates a positive effect on these phenotypes remains unclear and in order to clarify this different systematic approaches have simultaneously been followed. Random transposon mutagenesis and genomic bank screening allowed the identification a variety of genes that restore the swarming deficiency in an rsmA mutant when disrupted or when overexpressed. Among the clones obtained by screening the genomic bank exhibiting a restored swarming motility we discovered a previously unidentified open reading frame which encodes a 7.8 KDa protein sharing 34% identity and 52% similarity with the 6.9 KDa protein RsmA, which we termed RsmN. Although key residues important for RNA binding have been identified in RsmN based on its similarity with RsmA, this novel homologue presents some unique characteristics with respect to its domain architecture which seem to confer different specificities towards target mRNAs as the two proteins do not show total functional complementarity. Preliminary comparative analysis of RsmA and RsmN are presented and the possible implications of these results in the Rsm regulatory network model in P. aeruginosa discussed.


C-type natriuretic peptide (CNP) enhances quorum sensing factors production through activation of intracellular cyclic nucleotides synthesisWilfried Veron1, Anne-Sophie Blier1, Laure Taupin2, Karine Rehel2, Nicole Orange1, Marc GJ Feuilloley1 and Olivier Lesouhaitier1

1Laboratory of Cold Microbiology - Signals and Microenvironment, UPRES EA 4312, University of Rouen, Evreux, France, 2Laboratoire de Biotechnologie et Chimie Marines, Université de Bretagne-Sud, Lorient, France.

There is now ample evidence that eukaryotic signal molecules released by the host can modulate the virulence of prokaryotes, including opportunistic pathogens. We have previously shown that Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP) enhance

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Pseudomonas necrotic activity through activation of a cyclase and stimulation of the Vfr global regulator. In order to go further into the mechanism of action of BNP and CNP on Pseudomonas we investigated the effects of these eukaryotic peptides on quorum sensing factors in P. aeruginosa PAO1.Exposure of P. aeruginosa PAO1 to CNP (10-6 M) increased significantly the production of N-butanoyl homoserine lactone (C4-HSL). Conversely, the amounts of 2-heptyl-3-hydroxy-4-quinolone (PQS) and its precursor, 2-heptyl-4-quinolone (HHQ), were significantly reduced by CNP treatment. The production rate of 2-nonyl-4-quinolone (HNQ), another quinolone synthesized from HHQ, was also decreased by CNP. We also noted a strong inhibition of the production of the phenazyne pyocyanin in bacteria exposed to CNP. The same series of experiments was realized using BNP (10-6 M). This peptide closely related to CNP was without effect on C4-HSL, PQS, HHQ, HNQ and pyocyanin synthesis. Using the strain PAO9002, a vfr null mutant of P. aeruginosa PAO1, we showed that the Vfr protein is required for mediation of the inhibitory effect of CNP on pyocyanin biosynthesis. Since, the Vfr protein is activated by cyclic adenosine monophosphate (cAMP), we have evaluated the effect of cell permeable stable analogs of cAMP and cyclic guanosine monophosphate (i.e. dbcAMP and 8BcGMP, 10-5 M) on quorum sensing molecules production in P. aeruginosa PAO1. We observed that these analogs of cyclic nucleotide monophosphates also modulate the formation of acyl-homoserine lactones. Moreover both dbcAMP and 8BcGMP reduce the PQS, HHQ and HNQ synthesis by P. aeruginosa. The inhibitory effect of CNP on pyocyanin production was also fully reproduced by use of dbcAMP and 8BcGMP.Taken together, these data reveal that in P. aeruginosa PAO1 the pro-virulent effect of CNP is relayed by an intra-bacterial signal transduction pathway supported by cyclic nucleotides and the Vfr protein. Activation of this pathway by CNP appears to have a dual and opposite effect on the acyl-homoserine lactones and PQS systems.


Trapping a quorum: Exploring the bacterial density requirement for cell-cell communicationAimee K. Wessel1, Jodi Connell1, Matthew R. Parsek2, Jason B. Shear1 and Marvin Whiteley1

1University of Texas at Austin, 2 University of Washington, Seattle, USA

Many bacteria control group behavior using small signaling molecules in a process called quorum sensing. P. aeruginosa produces at least 4 cell-signaling molecules that control gene expression. It has been proposed that quorum sensing is mediated not only by cell density, but also by the spatial distribution of bacterial cells and flux through the environment. To test these hypotheses, we present a new methodology that allows for the monitoring of quorum sensing, while varying cell density, spatial distribution, and system flux. Dynamic masking multiphoton lithography techniques allow us to rapidly prototype various porous protein microstructures with measurable volumes on the low picoliter range. Upon inoculation, P. aeruginosa enter through a small opening of a protein microstructure. Once inside, the temperature is raised to 37 degrees Celsius and the entrance to the microstructure swells shut, thus constricting the opening and blocking exit of the bacteria. With a constant heated flow of nutrients, bacteria are trapped in small three-dimensional spaces, measuring between 5 to 20 um in length and width, and 8 um tall. Bacteria enclosed within these bacterial ''lobster traps'' remained motile and consistently doubled, increasing cell density in a confined space. Eventually, cell mass filled the trap volume. With this technology, the quorum sensing response can be monitored in populations possessing constant densities, but varying cell numbers. Quorum sensing gene expression was monitored in the traps using a P. aeruginosa strain carrying an rsaL-gfp fusion, and this fusion was shown to be responsive to exogenous 3-oxo-C12-HSL addition using confocal and epifluorescence microscopy. By varying the flow rate of fresh media and the size of the bacterial traps, we can directly monitor how flux, spatial distribution, and cell density/number contribute to quorum sensing.


Reconfiguring the quorum-sensing regulator SdiA of Escherichia coli to control biofilm formation via indole and N-acylhomoserine lactonesThomas Keith Wood1, Jintae Lee1, Toshinari Maeda1 and Seok Hoon Hong1

1Texas A & M University, USA

Previously, we developed the first synthetic signaling circuit to control biofilm formation (BMC Microbiol. 7:42, 2007) and engineered the first biofilm to reduce corrosion (J. Industr. Microbiol. Biotechnol. 22:167,1999). To control biofilms containing multiple species for engineering applications through the use of extracellular signals, here we have reconfigured the quorum-sensing regulator SdiA. We have shown SdiA interacts with the extracellular signal indole to reduce biofilm formation (BMC Microbiol. 7:42, 2007) primarily at low temperatures (ISME J. 2:1007, 2008). After random mutagenesis, we screened 4577 mutants with indole and two acylhomoserine lactones (AHLs) and identified four SdiA variants with altered biofilm formation including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and K153X) and SdiA14C3 (W9R, P49T, N87T, frame shift at N96, and L139X) which reduced biofilm formation by an additional 5- to 20-fold compared to wild-type SdiA. Whole-transcriptome profiling revealed that wild-type SdiA reduced biofilm formation by repressing genes related to indole synthesis, curli synthesis, and AI-2 uptake while SdiA1E11 induced genes related to indole synthesis, AI-2 uptake, and repressed genes related to cold-shock proteins. These results suggested altered indole metabolism, and corroborating the DNA microarray results in regard to indole synthesis, variant SdiA1E11 produced 9-fold more indole which led to reduced swimming motility and cell density. Also, wild-type SdiA decreased curli production and tnaA transcription while SdiA1E11 increased tnaA transcription (tnaA encodes tryptophanase which forms indole) compared to wild-type SdiA. Hence, wild-type SdiA decreased biofilm formation by reducing curli production and motility, and SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive variant, SdiA2D10 (E31G, Y42F, R116H, and L165Q), increased biofilm formation 7-fold in the presence of C8-AHL and oxo-C12-AHL. Therefore, SdiA can be evolved to increase or decrease biofilm formation, and biofilm formation may be controlled by altering sensors rather than signals.


Stereospecificity of pyochelin signaling in Pseudomonas aeruginosa PAO1 is conferred by the AraC-type regulator PchRZeb Youard1 and Cornelia Reimmann1

1University of Lausanne, Switzerland

Pyochelin is a thiazoline siderophore produced by Pseudomonas aeruginosa, Burkholderia cepacia and some strains of Pseudomonas fluorescens in response to iron limitation. Pyochelin is released into the extracellular environment, where it chelates iron and transports

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it back into the cell via a specific outer membrane receptor. Pyochelin upregulates its own biosynthesis and uptake genes by binding to and activating the AraC-type transcriptional regulator PchR in a process termed pyochelin signaling.Pseudomonas fluorescens CHA0 produces an enantiomer (non-superimposable mirror image) of pyochelin termed enantio-pyochelin. Enantio-pyochelin promotes growth under iron limitation in strain CHA0 and induces the expression of its biosynthetic genes, but is not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promotes growth and induces pyochelin gene expression in P. aeruginosa, but is not functional in P. fluorescens. In this study we investigated the role of PchR and the pyochelin/enantio-pyochelin receptors in stereospecificity of signaling. Using receptor and pchR mutant strains of P. aeruginosa and P. fluorescens we found that although the receptor is required for pyochelin/enantio-pyochelin mediated iron uptake, it is not an essential requirement for signaling. We also found that PchR is highly stereospecific and that its N-terminal domain is responsible for siderophore binding/recognition.

Pseudomonads as Pathogens

Oral presentations

O-14 Removed by request of the author before online publication.

O-15 Topic: Pseudomonads as Pathogens

The many faces of Pseudomonas aeruginosa in chronic obstructive pulmonary diseaseTimothy F. Murphy1

1University at Buffalo, The State University of New York, USA

Bacterial infection plays an important role in the course and pathogenesis of chronic obstructive pulmonary disease. Pseudomonas aeruginosa is isolated in sputum cultures from adults with COPD but the significance of the organism in this clinical setting is not well known. Because most studies to date have been cross sectional, it has been difficult to determine whether the bacterium is simply a colonizer or whether chronic infection occurs in adults with COPD, similar to that seen in the setting of cystic fibrosis. In a recent 10 year longitudinal study, two distinct patterns of carriage of P. aeruginosa are observed in adults with COPD: 1) short term colonization followed by clearance and 2) long term persistence. Acquisition of P. aeruginosa is associated with the occurrence of an exacerbation, indicating that the organism causes exacerbations. Exacerbations caused by P. aeruginosa are more likely to be seen in patients with more advanced COPD, those who have received recent antibiotic therapy and those who require mechanical ventilation for an exacerbation. Analysis of sputum samples by PCR reveals that P. aeruginosa is present far more often than indicated by bacterial culture. A subset of adults with COPD become chronically colonized with P. aeruginosa, but whether such patients benefit from antimicrobial therapy is not yet known.Genotyping of prospectively collected isolates from adults with COPD using a custom made microarray revealed that world wide dominant clones were also the most abundant clones among the COPD isolates. No prominent COPD-associated clones were detected.P. aeruginosa has several different manifestations in the setting of COPD. The organism is a colonizer that is cleared quickly, causes acute exacerbations and also may cause chronic infection in a subset of adults with COPD.

O-16 Topic: Pseudomonads as Pathogens

A virulence factor of the plant pathogen P. syringae pv. syringae is a preoteasome inhibitorRobert Dudler1, André Bachmann2, Michael Groll3, Markus Kaiser4, Steven Lindow5 and Barbara Schellenberg6

1University of Zurich, Switzerland, 2Cancer Res. Center, Univ. of Hawaii, Honolulu, HI 96813, USA, 3Inst. of Biochemistry, TU Munich, D-85747 Garching, Germany, 4Chemical Genomics Center, Max Planck Society, D-44227 Dortmund,Germany 5Dept. of Plant and Microbial Biology, UC Berkeley, CA 94720-3102, USA, 6Inst. of Plant Biology, Univ. of Zurich, CH-8008 Zurich, Switzerland

Syringolin A (SylA) is a structurally unusual peptide derivative produced by Pseudomonas syringae pv. syringae under in planta conditions. It is the product of a mixed non-ribosomal peptide/polyketide synthetase. Cloning of the SylA synthetase genes has allowed to generate mutants not producing SylA by gene disruption. Such mutants exhibit greatly reduced virulence on host plants (bean, Phaseolus vulgaris), indicating that SylA is a virulence factor. We have identified the eukaryotic proteasome as the target of SylA. The crystal structure of SylA in complex with the yeast 20S proteasome revealed that SylA covalently binds to all three catalytic subunits of eukaryotic proteasomes by a novel mechanism. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales, for which we demonstrate a similar proteasome inhibition mechanism. Because proteasome inhibitors are a new promising class of anti-tumour agents, the discovery of a new structural inhibitor class may also have medical implications. Genes with an architecture similar to SylA/GlbA synthetase genes are found in a small but intriguing group of pathogenic bacteria that are therefore hypothesized to produce SylA/GlbA-like proteasome inhibitors.


PO-149 Topic: Pseudomonads as Pathogens

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The pathogenicity and regulated production of the membrane vesicle from Pseudomonas aeruginosaJun Fukushima1, Shizuka Saito2, Taichiro Tanikawa2, Yoichiro Shimura2 and Tamio Inamoto2

1Laboratory of Microbiology, Akita Prefectural University, 2Laboratory of Microbiology, Akita Prefectural University, 241-438 Shimoshinjyo-Nakano, Akita 010-0195, Japan

The opportunistic pathogen P. aeruginosa has been known as a producer of small particles called membrane vesicles in the culture medium. The vesicle contains lipopolysaccharides, DNA, proteins and Pseudomonas quinolone signal (PQS) for quorum sensing. From these observations, we assumed that the membrane vesicle should be the important pathogenic factor. To assess this possibility, firstly we purify the membrane vesicle from the culture supernatant using density-gradient ultracentrifugation. Then the pathogenic activity was determined by using the human lung cancer cell-line, NCI-H292 with a mucin over-producing activity. The purified membrane vesicles are capable of inducing the mucin production in a concentration-dependent manner, and this induction was partly inhibited by adding Azithromycin, a member of the macrolide antibiotics, represents the membrane vesicle has a mucin-induction activity and which should be inhibited by the macrolide. We next investigated the regulated expression of membrane vesicles from P. aeruginosa. The PQS has been reported as an essential factor for vesicle production. We determined the role of upstream factors for biosynthesis of PQS molecules, such as classical Quorum sensing system using acyl-homoserine lactone molecule as an autoinducer for the membrane vesicle-production. We have used the mutant of lasI and rhlI that are the genes for biosynthesis of acyl-homoserine lactone molecules in P. aeruginosa. The produced vesicle amounts were determined using OD measurements at 220 nm of the precipitated fraction of culture supernatant using ultracentrifugation. The mutants could not produce membrane vesicles, and were complemented by the wild-type gene that was introduced into each mutant and also by adding the corresponding autoinducer. From these data, we confirmed that both autoinducers are required for production of the membrane vesicles. In this case, we also added the Azithromycin to the culture supernatant of wild type strain at the sub-MIC level to see the effect on the vesicle formation because the macrolide have been reported as an inhibitor of Quorum sensing. The amount of vesicles were decreased according to the concentration of Azitromycin showing that Azithromycin inhibits the vesicle production in P. aeruginosa. We speculate that the Azithromycin affects both the host cells and the bacterial cells, and this effect should be one of the mechanisms of Azythromycin treatment against chronic infection of P. aeruginosa.


Identification of Factors Involved in Melanin Overproduction within the Cystic FIbrosis LungRyan C Hunter1 and Dianne Newman1

1Massachusetts Institute of Technology Boston, USA

Melanin overproduction is a common phenotype amongst Pseudomonas aeruginosa isolates from cystic fibrosis infections. In addition, melanogenic variants of strain PA14 arise within in vitro biofilm communities, suggesting that pyomelanin production confers a competitive advantage to the producing strain or the entire biofilm community. Possible roles for melanin include iron acquisition, protection from oxidative burst, antimicrobial tolerance and electron shuttling, but the precise role of this pigment in the context of CF infection remains unclear. Regardless of function, there is still a need for a better understanding of the environmental and molecular factors that lead to its production. In this study, we undertook a transposon mutagenesis approach to elucidate gene products involved in P. aeruginosa melanogenesis. 13,000 mutants of a melanin overproducing clinical isolate were screened for a decrease in reddish-brown pigmentation. Arbitrary PCR led to the identification of 22 mutants with mini-mariner transposon insertions in genes whose products are involved in amino acid catabolism, nucleotide biosynthesis, two-component regulatory systems, membrane transport, and hypothetical proteins with unknown function. One mutant, D741, had a transposon insertion in a gene sequence predicted to encode an ABC-efflux transporter with homology to a resistance-nodulation-division protein involved in organic solvent tolerance in P. putida. Given the structural similarity between toluene and homogentisic acid (the tyrosine metabolism precursor of pyomelanin), it is possible that this gene product facilitates homogentisate export into the surrounding medium. Growth assays and further characterization of this mutant by HPLC showed a reduced production of melanin in the extracellular space over time, with a corresponding accumulation of homogentisate within the cell, which supports a role for this putative ABC efflux transporter in P. aeruginosa melanin production. Interestingly, a bioinformatics approach identified homologs of this protein in other bacterial species known to produce melanin in CF lung infections (Burkholderia cepecia, Stenotrophomonas maltophila). Further characterization of factors which lead to the overproduction of melanin may ultimately lead to novel targets for antimicrobial strategies.


Snatch of the host immunity by quorum-dependently secreted factors from Pseudomonas aeruginosaJoon-Hee Lee1, Su-Jin Park1, Ji-Won Park2, Yu Sang Choi1, Soo Kyoung Kim1 and Bok-Luel Lee2

1Lab of Microbiology, College of Pharmacy, Pusan National University, Busan, 609-735, South Korea, 2National Research Lab. of Defense Proteins, College of Pharmacy, Pusan National University, Busan, 609-735, South Korea

Invertebrates mainly depend on innate immunity for defense against pathogenic bacteria. These defenses include the expression of antibacterial peptides and the activation of prophenoloxidase cascades in hemolymph (insect blood). Generally, these systems are initiated from the recognition of bacteria-specific materials, so called PAMPs (pathogen-associated molecular pattern) such as peptidoglycan, lipopolysaccharide, lipoteichoic acid, and glucan by the cognate specific recognition proteins (pattern recognition receptors). In insects, some of these recognition signals are amplified by a proteolytic cascade similar to the vertebrate complement system, which activates the Tol-mediated intracellular signaling pathway to produce antibacterial peptides. In hemolymph, the prophenoloxidase activation leads to melanization on the infected site to block the further infection process, which is another major innate immune defense mechanism in invertebrates. While these pathways are specifically cascaded by host protease system, bacteria also produce many proteases during the infection process, which are known as virulence factors. A multi-host pathogen, Pseudomonas aeruginosa produces many extracellular proteases involved in pathogenecity, including elastase (pseudolysin), alkaline protease (aeruginolysin), LasA (staphylolysin), LasD (staphylolysin), and Protease IV (lysyl endopeptidase). In this study, we investigated the possibility that bacterial proteases could lead the activation or interrupt of the insect innate immune system by cleaving the host proteases. When the cell-free culture supernatant (CFCS) of P. aeruginosa was injected into Tenebrio moliter, the melanization was induced. However, the small molecules passed through the 10 kDa cut-off membrane or the heat-inactivated CFCS failed to induce the melanization. Interestingly, CFCS from quorum mutant (lasI-, rhlI-) also failed to induce the melanization and it was restored by the supplement of synthetic quorum signals during the culture. This suggested that certain quorum-dependently secreted protein factor(s) may snatch the innate immune response, skipping the initial recognition step. When we mixed the CFCS with the purified protease cascade components of the T. moliter innate immune system in vitro, some of them were significantly cleaved into their active forms.

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Here, we explored the bacterial proteases involved in this process and addressed the role of quorum sensing in the Pseudomonas infection.


A role for cyclic-di-GMP in persistent Pseudomonas aeruginosa infectionsJacob Malone1, Tina Jaeger2, Regine Landmann3 and Urs Jenal2

1University of Basel, 2Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland, 3University Hospital Basel, University of Basel, Hebelstrasse 32, 4031 Basel, Switzerland

Pseudomonas aeruginosa small colony variants (SCVs) are distinctive morphotypes, routinely isolated from the lungs of cystic fibrosis patients, whose appearance correlates with poor lung function and persistence of infection. Accumulating evidence links the SCV morphotype to the second messenger cyclic-di-GMP. The yfiBNR operon was identified as a key SCV regulator. This operon encodes a cyclic-di-GMP generating enzyme (DGC, YfiN), an outer membrane protein (YfiB), and a small periplasmic protein (YfiR). Here we propose a model for YfiBNR function, whereby YfiN acts as a membrane-integral DGC whose activity is controlled through YfiR and YfiB. A secondary genetic screen identified exopolysaccharide synthesis as the primary downstream target for YfiBNR, with disruption of the pel and psl operons leading to suppression of the delta-yfiR SCV phenotype.Delta-yfiR was subsequently used as a representative SCV in cell culture and animal studies of persistent infection. The delta-yfiR SCV exhibited strong resistance to macrophage absorption compared to wild-type, and effectively persisted in mouse infection models despite exhibiting a marked disadvantage in vitro. Together, these results establish YfiBNR as an important player in persistence of P. aeruginosa and implicate a central role for c-di-GMP in chronic infections.


Pseudomonas aeruginosa pathogenisis in zebrafish embryosMarlies J. Mooij1, Astrid van der Sar2, Marian Llamas2, Claire Adams1, Wilbert Bitter2 and Fergal O'Gara1

1University College Cork, Ireland, 2VU University medical center, The Netherlands

Pseudomonas aeruginosa has been reported to cause disease in plants, insects, and a variety of vertebrates. The plant Arabidopsis thaliana and the soil nematode Caenorhabditis elegans have both been used to identify virulence-related genes of this pathogen. Strains harboring mutations in most of these genes were also attenuated in virulence when analyzed in a murine infection model, supporting the utility of other models than mammalian systems. In the last years, zebrafish has been recognized as a useful model to study bacterial infections in real time. In this study we investigated the pathogenesis of P. aeruginosa in zebrafish.For in vivo monitoring of this pathogen, we used P. aeruginosa expressing the DsRed variant mCherry. We exposed zebrafish embryos to ~109 CFU/ml P. aeruginosa strain PAO1 by static immersion. Red fluorescence was observed in the intestines and faeces of the embryos. Two days after infection, about 80 to 100% of the embryos died, and mortality was characterized by necrosis of the tissue. Unexpectedly, heat-killed P. aeruginosa was also capable of killing the zebrafish embryos. This is thought to be due to presence of a heat-stable toxin, of which the nature will be further investigated.When zebrafish embryos were infected by micro-injection with a low dose of PAO1 (~200 CFU), mortality rates up to 60% (5 days post infection) were observed. A high dose of PAO1 (~2000 CFU) is lethal for the embryos within 1-2 days. Subsequently, we tested P. aeruginosa mutants affected in genes known to be essential for virulence, including the quorum sensing lasIR genes, the response regulator gacA, the xcp type II secretion system genes, or the type III secreted exoenzymes exoS, exoT and exoY. Virulence of these mutants was not attenuated in 24 hpf embryos, but this will be repeated in older zebrafish embyros to verify if developmental stages of the embyro plays a role in infection.Matrix metalloproteases (MMPs) are proteolytic enzymes that regulate extracellular matrix turnover and aid in restoring tissue architecture following injury. Microarray analysis of zebrafish embryos upon micro-injection with P. aeruginosa showed upregulation of the matrix metallo-proteases, MMP-9 and MMP-13. Proteolytic dysregulation plays a role in chronic neutrophilic lung diseases, such as cystic fibrosis, since increased levels of MMP-9 were detected in sputa from cystic fibrosis patients. The nature of MMP-9 and MMP-13 induction will be further investigated.


Phenotypic diversity and interactions between Pseudomonas aeruginosa CF isolates from individual sputum samplesDouglas Gordon Storey1, Erika Lutter2, Monica Faria2, Justin Wong2, Jen Amon2 and Harvey Rabin3

1University of Calgary, 2University of Calgary, Biological Sciences, 3University of Calgary, Medicine, Microbiology and Infectious Diseases, Canada

P. aeruginosa isolates from CF patients are known to be genotypically and phenotypically diverse. However, the level of diversity of P. aeruginosa within a single patient, at any one time point is only beginning to be appreciated. A survey of bacterial populations from 7 CF patients showed that these populations had both genotypic and phenotypic diversity. These patients were all adult CF patients who had been colonized for years and so the phenotypic diversity suggested that the sub-populations of P. aeruginosa isolates may be working together in a cooperative manner to maintain the infection. We have examined the interactions between the isolates from each patient using the Drosophila feeding infection model to look at mixed infections. Interestingly, one pair of isolates had an additive effect with respect to lethality in this model system. However, two different pairs of isolates had a profound negative effect on lethality in the fly model. We have further characterized these negative interactions and showed that a secreted molecule was responsible for this effect. Further, this negative interaction appeared to influence the production of proteases in susceptible cells. An analysis of this effect on other CF isolates showed that 8 CF isolates from 5 additional patients could respond to the secreted factor. Finally, we showed that closely related P. aeruginosa isolates to the producer isolate, were not affected by this signalling molecule whereas a group of less related isolates did respond to this negative signal. Overall, these interactions may suggest a mechanism for maintaining a chronic infection in the lungs of patients with CF where protease and perhaps other virulence factors are attenuated in one sub-population by another member of the bacterial population.

PO-155

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Topic: Pseudomonads as Pathogens

Characterization of a new secondary metabolite that plays a key role in Pseudomonas entomophila virulence towards Drosophila melanogasterIsabelle Vallet-Gely1, Onya Opota2, Alexey Novikov3, Eric Guittet4, Frederic Boccard5 and Bruno Lemaitre2

1CNRS, 2EPFL Lausanne, 3IBBMC Orsay, 4ICSN Gif sur Yvette, 5CGM Gif sur Yvette, France

We developed a new model to study host-pathogen interactions, using Drosophila as the host and Pseudomonas entomophila as the pathogen. In contrast to other models involving Drosophila, which require the injection of bacteria directly into its body cavity, our model relies on P. entomophila virulence after oral ingestion, a more natural route of infection.P. entomophila has the capacity to induce the systemic production of antimicrobial peptides, and it is highly pathogenic to drosophila, as well as to other insects. Its persistence in the insect gut leads to a massive destruction of epithelial cells.The complete sequence of the P. entomophila genome was determined, and compared to the other sequenced Pseudomonas genome. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather seems to rely on a number of potential virulence factors to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that could regulate most of the potential virulence factors identified.Using a combined genetic approach on both host and pathogen, we started to explore the contribution of these multiple factors to the infectious process. Here we describe in further detail the characterization of a new secondary metabolite that plays a key role in the infectious process. Its structure was elucidated using NMR, and several genes are involved in its synthesis, which is regulated by the GacS/GacA two component system. Mutants unable to synthesize this secondary metabolite are affected in their abilities to persist in the fly gut, to trigger antimicrobial peptide production and to efficiently kill Drosophila.


Virulence factors associated with the sessile lifestyleMartin Welch1, Kahoko Nishikawa2, Kathryn Lilley3, Helga Mikkelsen4 and Jade Chung3

1Cambridge University, UK, 2Department of Traumatology and Critical Care Medicine, National Defense Medical College, 3-2 Namiki Tokorozawa, Saitama, 359-9513 Japan, 3Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK, 4Centre for Molecular Microbiology and Infection Division of Cell and Molecular Biology Faculty of Natural Science Flowers Building, 1st floor South Kensington Campus Imperial College London London SW7 2AZ, UK

We have used quantitative proteomic analysis to analyse the secreted proteins produced by Pseudomonas aeruginosa biofilms, and compared these with the spectrum of proteins secreted by planktonic cells. The protein profile associated with each growth mode is clearly different. In particular, we found that biofilms (but not planktonic cells) of P. aeruginosa express the Type 3 secretion (T3S) machinery and also secrete T3S system-dependent exotoxins. Microarray analyses confirmed that the T3S system genes were up-regulated in these biofilms. Subsequent work has shown that expression of the T3SS correlates strongly with anaerobiosis. Indeed, planktonic cultures of PAO1 can be made to up-regulate expression of the T3S machinery in anaerobic conditions. Interestingly, we found that in these growth conditions, the profile of N-acyl homoserine lactones (AHL) produced by PAO1 is inverted compared with aerobic cultures, such that BHL is the dominant AHL species. This is reminiscent of the AHL profile previously reported for CF sputum and is relevant to T3S because AHLs have been shown to affect the expression of this secretion apparatus. These findings, along with more recent ones focussing on the explicit link(s) between T3S, quorum sensing and anaerobiosis will be discussed. In addition, we have found that sessile cells (biofilms and colonies), but not planktonic cultures of PAO1 express a pair of uncharacterized proteins; PA2782 and PA2783. These genes appear to be highly expressed in young (developing) biofilms, and expression declines with time, suggesting that they may play a role in biofilm establishment. PA2783 was experimentally demonstrated to be a secreted protein, and bioinformatic analyses show that it is a metzincin-class protease with most similarity to the proteases present in rattlesnake venom. Unlike the only other metzincin encoded by P. aeruginosa (AprA, alkaline protease) which is found in all Pseudomonads sequenced to date, PA2782/3 are only found in P. aeruginosa, P. mendocina and P. putida, indicating a more specialized, pathogen-specific role for these gene products. PA2783 carries a pair of carbohydrate-binding domains, which presumably enables the protein to be targeted to specific sites, either in the host or in the developing biofilm. The secretion pathway employed by PA2783, the possible biological activities/role(s) of PA2782/3 and the expression profile(s) of these proteins in CF sputa will be presented.

P-157 Topic: Pseudomonads as Pathogens

Comparison of Pseudomonas aeruginosa gene expression across multiple in vivo infection conditions Piotr Bielecki1, Jacek Puchalka1, Melissa Wos2, Holger Loessner3, Justyna Glik4, Marek Kawecki4, Siegfreid Weiss3, Kenneth Timmis5, Burkhard Tümmler6 and Vítor AP Martins dos Santos1

1Systems and Synthetic Biology Group, Helmholtz Centre for Infection Research (HZI) Braunschweig, Germany, 2Biodegradation Research Group, HZI, Braunschweig, Germany 3Molecular Immunology, HZI, Braunschweig, Germany 4Centrum Leczenia Oparzen. Siemianowice Śląskie, Poland, 5Environmental Microbiology Group, HZI, Braunschweig, Germany 6Klinische Forschergruppe, Medizinische Hochschule Hannover, Germany

Pseudomonas aeruginosa is a major opportunistic pathogen causing nosocomial infections in immunocompromised patients such as burn wound victims. It is also a major threat for cystic fibrosis patients. Data on the molecular mechanisms underlying infection by P. aeruginosa have been derived from numerous in vitro and ex vivo studies, but there are very few reports on in vivo infection data. To gain insights into the combinatorial pathogenicity of this threatening pathogen upon infection and specificity of virulence factors in different infections, we measured for the first time the in vivo gene expression profiles of P. aeruginosa during burn wound infections and compared them with other in vivo infection settings, namely plant infection and mouse tumor infection as well as with in vitro conditions comprising of planktonic and biofilm growth on abiotic surface. The P. aeruginosa strains used in this study are three distinct clones isolated from human burn wounds. Using multivariate statistical analysis we observed that these clones are clustered together within each infection setting, which suggests that the site-specific conditions more strongly influence global expression than clone-specific genetic composition. Multidimensional scaling showed the pattern of similarities and dissimilarities, where burn wound infection was significantly different from control conditions as well as model infection conditions. Those results underscore the previously developed hypothesis that P. aeruginosa infections are of multifactorial and combinatorial nature, whereby strains have a pool of pathogenicity-related traits that interact in different combinations against the different host backgrounds. Therefore, infection models may be used only to limited extent. Among the genes expressed exclusively in burn wound infection, we can distinguish known

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virulence factors such as Exotoxin A regulator and as well yet uncharacterized systems presumably involved in iron acquisition, secretion, gas exchange and DNA repair. Pairwise comparisons of burn wound infection with control conditions proved the importance of haem and zinc acquisition in infection. Furthermore, the analysis has enabled us to pin-point a number of genes encoding conserved hypothetical membrane proteins and transport systems that may be promising candidates for further functional analysis in the context of infection.


Genes influenced by the Lon protease of Pseudomonas aeruginosa and its role in pathogenesisElena B.M. Breidenstein1, Manjeet Bains2, Patrick K. Taylor2 and Robert E.W. Hancock2

1University of British Columbia, 2University of British Columbia, Department of Microbiology and Immunology, Vancouver, British Columbia, V6T1Z4, Canada

Background: The ATP-dependent Lon protease of Pseudomonas aeruginosa PAO1 is involved in pathogenesis and resistance to ciprofloxacin and has an 84% similarity to the Lon protease of Escherichia coli. Studies from our lab have previously shown that the Lon protease is involved in resistance to ciprofloxacin, motility (swarming and twitching) and biofilm formation. Methods: Microarray analyses were performed to investigate differentially expressed genes in the PAO1 transposon-inserted lon mutant (mid-log and stationary phase) compared to the wild type PAO1, as well as in sub-inhibitory concentrations of ciprofloxacin. The RNA from each treatment was isolated, reverse transcribed into cDNA and then labeled with cy3 or cy5 dyes. Labeled sample pairs were applied to a spotted microarray, hybridized and scanned on a ScanArray Express scanner and analyzed using Imagene software and ArrayPipe version 1.7. To validate the microarrays, several genes were confirmed by real-time PCR. Chemotaxis assays were performed on soft agar plates with different carbon sources. An LDH-assay was used to measure cytotoxicity over 4 and 10 hours for the lon mutant and the wild type. Results: Several genes belonging to the functional classes of motility and attachment, energy metabolism and secreted factors were down-regulated in the lon mutant. This explained the observed motility defect of the mutant, as well as the defect in cytotoxicity that was newly detected. Genes involved in chemotaxis were not differentially expressed in the lon mutant, indicating that lon mutants should show normal chemotaxis behavior. Consistent with this the lon mutant exhibits normal chemotaxis to a variety of carbon sources. Investigation of differentially expressed regulators indicated that these formed a regulatory hierarchy. To follow up on the role of Lon protease in pathogenesis and to determine which proteins were cleaved by the Lon protease, a lon deletion knockout mutant is being constructed. Conclusion: These results reinforce the assumption that the Lon protease plays an important role in pathogenesis of P. aeruginosa. Furthermore, the microarray analyses allow us to clearly determine which genes were differentially expressed in the lon mutant and which of these were influenced by ciprofloxacin as well as those genes that were only differentially expressed in the lon mutant in the presence of ciprofloxacin.


Identifcation of P. aeruginosa in vivo essential genes in zebrafishNikhilesh Singh Chand1, Anne Clatworthy2, Jenny Lee2, Toshiro Ohsumi2, Mark Borowsky2 and Deborah Hung3

1Harvard University, Massachusetts General Hospital, 2Massachusetts General Hospital, 3Massachusetts General Hospital, Broad Institute, USA

In vivo essential genes may encode proteins necessary for colonization, virulence factors or metabolic proteins critical for survival within a host. An in vivo essential gene may not be critical in vitro, while a gene that appears important in an in vitro assay may not be required in vivo. Identifying bacterial genes essential in vivo can lead to the discovery of novel virulence determinants and potential antibacterial drug targets. In vivo essential genes can be identified by screening for mutants attenuated for infection in animal models individually, which is laborious or by screening complex mutant pools using a negative selection strategy such as TraSH (Transposon-site hybridization). We have used a slightly modified TraSH approach to screen pools of PA14 transposon mutants for in vivo essentiality in zebrafish embryos, a recently characterized host model that closely resembles rodent models of acute Pseudomonas infection. Since Pseudomonas aeruginosa has the most extensive set of two-component systems (approximately 64) known in a single organism, we first screened a small pool of transposon mutants comprised of annotated two-component systems from the PA14 non-redundant transposon library for essentiality in zebrafish embryos. Several transposon mutants, including two uncharacterized two-component regulators were found to be attenuated when tested individually in zebrafish embryos, suggesting that the disrupted region is important for viability in vivo. In parallel to examining the essentiality of two-component systems in zebrafish embryos we are also developing a negative selection strategy that relies on Illumina DNA sequencing rather than microarray hybridization to identifiy in vivo essential genes. This offers the advantage of less false positives and negatives and circumvents the need for a PA14 array. We plan to use this strategy to screen the entire non-redundant transposon library for genes essential in vivo not only in zebrafish but also in other rodent models. This would allow us to compare genes essential for viability in a diverse set of vertebrate host models.


Aerobic desaturase (DesB) is important for virulence of Pseudomonas aeruginosa PAO1Kyoung-Hee Choi1 and Herbert Schweizer2

1Wonkwang University, Iksan, South Korea, 2Colorado State University, Fort Collins, USA

Unsaturated fatty acids (UFAs) play a vital role in regulating the fluidity of bacterial membranes. UFA levels are up- or down-regulated in response to various environmental conditions to ensure maintenance of appropriate bacterial membrane fluidity, which in turn affects numerous cellular functions. The synthesis of UFAs in P. aeruginosa is governed by two pathways: 1) the anaerobic FabAB-mediated pathway and 2) the aerobic inducible desaturase pathway. There are two distinct desaturase genes in P. aeruginosa, desA encoding acyl-lipid desaturase and desB encoding acyl-CoA desaturase. The desB gene forms an operon with desC, encoding a putative oxidoreductase, and the desBC operon is under DesT repressor control.Previously, it was suggested that bacterial desaturases (eg. DesA1 and DesA3 in Mycobacterium sp.) are required for full virulence and thought to be novel therapeutic targets. Based on the previous findings that desaturase proteins play an essential role in bacterial virulence, we characterized the relationship between P. aeruginosa desaturase proteins and virulence. We used the following mutants: fabA, desA, desB and desT single mutants, fabA desA and fabA desB double mutants, and a fabA desA desB triple mutant. A fabA mutant is defective in UFA biosynthesis, while the desA desB double mutant lacks the enzyme activity for desaturating saturated fatty acids. Using these mutants we investigated production of various important virulence factors. Virulence factors tested include the production

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of pyocyanin pigment, total protease and elastase, rhamnolipids, and motilities (swarming, swimming and twitching). Furthermore, in vivo virulence was assayed using Caenorhabditis elegans nematodes. Testing of individual mutations or combinations of mutations revealed that desB mutants were severely deficient in proteolytic enzyme, pyocyanin and rhamnolipid production. In addition, the desB mutants were impaired in swarming and twitching motilities, and showed reduced virulence in the C. elegans infection model. Taken together, these results demonstrate that desB is not simply a fatty acid desaturase but also required for full virulence in P. aeruginosa. The findings also suggest that DesB may be a novel drug target. However, further studies are needed to elucidate how DesB activity, UFA levels and/or membrane fluidity control expression and/or levels of the virulence factors examined in this study.


Type III secretion system in Pseudomonas aeruginosa biofilms.Jade CS Chung1 and Martin Welch2

1University of Cambridge, 2University of Cambridge, Hopkins Building, Tennis Court Road, Cambridge CB2 1QW, UK

We are investigating the association between anaerobiosis and the Type III secretion system (T3SS) in Pseudomonas aeruginosa (PA). This study follows from our recent published findings that T3S genes and proteins are up-regulated in PAO1 biofilms. Reports in the literature have shown that quorum sensing represses T3S. Since QS is known to be active in biofilms, T3S and biofilm formation are often thought to be mutually exclusive. However, further studies of PcrV expression, a core structural component and indicator of a functional T3SS, show that T3S also takes place in biofilms of at least one other virulent PA strain, PA14. Whilst similar studies are being conducted to identify T3S expression in biofilms of other highly virulent PA strains, it is clear that regulation of T3S in biofilms is not as straightforward as currently thought.These results, together with the observation that the expression of T3S and anaerobic respiration genes are highly correlated in PAO1 biofilms, led us to believe that there is a link between T3S and anaerobiosis. Indeed, we found that when planktonic PAO1 cultures were grown under anaerobic conditions, they exhibited higher PcrV expression compared to their aerobic counterparts. Furthermore, a double mutant in lasR and rhlR, quorum sensing regulators involved in the negative regulation of T3S, showed higher PcrV expression in both aerobic and anaerobic cultures as expected, but the increase was greater under anaerobic conditions.The influence of quorum sensing on T3S expression under anaerobic conditions was further investigated by examining the N-acyl homoserine lactone (AHL) profile of planktonic PAO1 cultures. Interestingly, C4-HSL (BHL) production is greatly enhanced during anaerobic planktonic growth. This result resembles the AHL profile previously reported for cystic fibrosis sputum. Likewise, analysis of AHL production in the effluent of PAO1 biofilms grown in a continuous flow system also indicates a significant up-regulation of BHL production from the second day of growth - despite the fact that T3S was shown to be active in this system. Together, these results suggest that quorum sensing plays a different role in regulating T3S under anaerobic conditions.Future work will investigate the genetic regulation of T3S under anaerobic conditions, as well as localization studies of T3S expression within the biofilm architecture.


Analysis of a new beta-lactamase involved in the virulence of P. aeruginosaAlicia Fajardo1, Pierre Cornelis2 and Jose Luis Martínez1

1Centro Nacional de Biotecnología, 2Vrije Universiteit. Brussels. Belgium

In hospitals, the capability of bacteria to produce infection relies not only in its virulence, but also in its low antibiotic susceptibility [1]. To analyze antibiotic resistance/bacterial virulence crosstalk, we have searched mutants simultaneously affected in both processes using a transposon-tagged library obtained in the P. aeruginosa 59.20 strain [2]. Altered susceptibility to one or more antibiotics was observed in 113 of the 2058 tested mutants (5,49%) [3]. To evaluate virulence, we have studied changes in their cytotoxicity against a macrophage cell line. The position of the transposon was determined by inverse polymerase chain reaction, sequencing of the amplicons, and comparison to the available sequence of P. aeruginosa (http://www.Pseudomonas.com). After filtering for insertion occurring in the same gene, we have detected 39 loci in the genome of P. aeruginosa 59.20 that contribute to changes in its virulence and antibiotic susceptibility simultaneously.We present in detail a study of one mutant, hyper-susceptible to imipenem and meropenem, and less cytotoxic than wild type strain.When we compared the amplified regions that flanked the transposon with the database of P. aeruginosa PAO1, we obtained that the transposon was situated in a gene that codes for a probable beta-lactamase. We made a deletion mutant and over expressed this gene in order to verify its phenotype of hyper-susceptibility and reduced cytotoxicity. Finally, we have identified its potential substrates by a biochemical assay. We can conclude: 1) the phenotype of virulence and antibiotic resistance in P. aeruginosa involves a complex network of elements, including non-classical antibiotic-resistance and virulence genes. 2) We have identified a new beta-lactamase in the chromosome of P. aeruginosa and its potential substrates. A mutation that inactives this gene makes this mutant less cytotoxic, so there is a crosstalk between resistance to beta-lactams and virulence in P. aeruginosa.[1] Martinez, J. L. & Baquero, F. Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol Rev 15, 647-79 (2002).[2] de Chial, M. et al. Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa. Microbiology 149, 821-31 (2003).[3] Fajardo, A. et al. The neglected intrinsic resistome of bacterial pathogens. PLoS ONE 3, e1619 (2008).


Genomic variations in a cystic fibrosis epidemic strain of Pseudomonas aeruginosa highlight the limitations of current diagnostic tests.Joanne L Fothergill1, Juliet E Foweraker2, Martin J Walshaw3, Martin J Ledson3 and Craig Winstanley4

1NIHR Biomedical Research Centre, Royal Liverpool & Broadgreen University Hospital, Liverpool, UK, 2Department of Microbiology, Papworth Hospital, Cambridge, UK., 3Regional Adult Cystic Fibrosis Centre, Cardiothoracic Centre, Liverpool, UK, 4Division of Medical Microbiology, University of Liverpool, UK

The transmissible Liverpool epidemic strain (LES) of Pseudomonas aeruginosa has been associated with increased morbidity in cystic fibrosis patients. Furthermore, this unusual strain has also been found in multiple locations in the UK, and North America. In Liverpool, up to 80% of CF patients infected with P. aeruginosa harbour the LES and identification of this strain leads to segregation of patients.

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In clinical laboratories, two typing methods are used to identify the LES, the published PCR tests (PS21 and LES-F9) and pulsed field gel electrophoresis (PFGE). Using these typing methods, 25 anomalous isolates from across the UK, were identified that gave inconsistent results with regards to either PFGE or PCR tests. The Clondiag tube array genomotyping system was used on these isolates to determine small nucleotide polymorphisms (SNPs) in the core genome of P. aeruginosa. This method enabled us to determine whether the isolates shared the same genomic backbone as the LES. Of the 25 anomalous isolates tested, 13 isolates were found to be the LES using this system. The genome sequenced LES isolate, LESB58 contains five genomic islands and five complete prophages. The presence of these prophages can vary between LES isolates, particularly during exacerbations. The presence/absence of prophages and selected genomic islands in the 25 anomalous isolates was determined and revealed differences in genomic content between the isolates. Variations in PFGE were often associated with the deletion of genomic islands and/or prophages. The rapidly evolving nature of the LES suggests that correct identification could be a challenging task for clinical laboratories in the future.This work was funded by the NIHR as part of the Biomedical Research Centre in Microbial Diseases.


Contributions of the PAPI-1 and PAPI-2 pathogenicity islands to the virulence of Pseudomonas aeruginosa strain PA14 in murine models of acute pneumonia and bacteraemia.Ewan M Harrison1, Melissa Carter 2, Aras Kadioglu2 and Kumar Rajakumar3

1Department of Infection, Immunity, and Inflammation, University of Leicester, Maurice Shock Building, University Road, University of Leciester, Leicester, LE1 9HN, 2Department of Infection, Immunity, and Inflammation, Maurice Shock Building University Road, University of Leicester Leicester, LE1 9HN, UK, 3Department of Infection, Immunity, and Inflammation, Maurice Shock Building University Road, University of Leicester Leicester, LE1 9HN, UK and Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester LE1 5WW, United Kingdom

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and severe, chronic lung infections in cystic fibrosis patients. P. aeruginosa strains PAO1 and PA14 have been used extensively as reference strains both in vitro and in vivo to study the virulence of this pathogen. PA14 has been found consistently to be more virulent than PAO1 in a number of diverse infection models. Among other factors, this difference in virulence has been suggested to be due to the presence of two pathogenicity islands, PAPI-1 and PAPI-2, both of which are present in PA14 but not in PAO1. In this study, we directly compared the global contributions of PAPI-1 and PAPI-2, rather than that of individual island-borne genes, to the virulence of PA14 using murine models of acute pneumonia and bacteraemia. We created three isogenic island-minus mutants (PA14dPAPI-1, PA14dPAPI-2 and PA14dPAPI-1dPAPI-2) and compared these with the wild-type parent strain PA14 and the less virulent PAO1. Our results showed that both pathogenicity islands contributed significantly to the virulence of PA14 in both models of acute pneumonia and bacteraemia. However, unlike the bacteraemia model where each island was found to contribute significantly individually to the virulence potential of PA14, loss of the large 108 kb PAPI-1 alone was insufficient to measurably attenuate the mutant in the acute pneumonia model. Nevertheless, the double mutant PA14dPAPI-1dPAPI-2 was substantially more attenuated in both models and exhibited a lesser degree of virulence than even PAO1 in the acute pneumonia model. In particular, the double mutant appeared to be markedly inhibited in its ability to disseminate from the lungs to the bloodstream in the pneumonia model. We conclude that both PAPI-1 and PAPI-2 contribute directly and synergistically in a major way to virulence of PA14 and suggest that analysis of island-minus strains, rather than individual gene knock-outs, may be a more appropriate way to assess the contribution to virulence of large, horizontally acquired segments of DNA.


The role of c-terminal processing peptidases in protein secretion and virulence of Pseudomonas aeruginosaRien Hoge1, Susanne Wilhelm1, Karl-Erich Jaeger1 and Frank Rosenau1

1 Heinrich-Heine-University Duesseldorf, Germany

Carboxy-terminal processing peptidases (CTPs) are periplasmic proteases that cleave a defined part from the C-terminus of certain proteins. In the genome of P. aeruginosa, the genes prc and ctpA were identified which encode CTP homologous proteins. P. aeruginosa secretes several virulence factors, thus, we have investigated the effects of Prc and CtpA on protein secretion and virulence.Secretion of extracellular proteins was investigated by comparing the supernatants of P. aeruginosa wild-type and ctpA and prc deficient mutants by 2D-gel electrophoresis and subsequent spot identification by MALDI-TOF peptide fingerprinting. The 2D-gel analysis showed significant differences in the secreted protein patterns. Several proteins were indentified that were present in decreased amounts in the deficient mutants, among them major virulence factors, namely elastase LasB, staphylolysin and protease IV. Also, several proteins were presented in increased amounts such as flagellin type B, flagellar capping protein D and an enterocheline esterase. Enzyme assays confirmed the proteomics results. The enzymatic activities of elastase, staphylolysin, protease IV and alkaline protease were all significantly decreased in culture supernatants from ctpA and prc deficient mutants.The Galleria mellonella caterpillar pathogen infection model was used to investigate virulence of the protease deficient mutants. Defined cell numbers of wild-type P. aeruginosa and ctpA and prc deficient mutants were injected to larvae of G. mellonella which were subsequently scored dead or live and LD50 values were calculated. Both protease deficient mutant strains showed a decline in virulence as compared to the wild-type strain.Finally, subcellular localisation studies revealed that CtpA is solely present in the periplasm of P. aeruginosa thereby confirming its hypothetical localization. The ctpA gene was cloned into vector pIX3.0 and successfully expressed by in vitro transcription using E. coli cell lysates. The purified protein showed protease activity as determined with beta-casein as the substrate.


Involvement of KatA in virulence is associated with aerobic NO accumulation in Pseudomonas aeruginosaSeol-Hee Kim1, Min-Gyeong Kang1 and You-Hee Cho1


KatA, the major catalase of Pseudomonas aeruginosa strain PA14 is a critical virulence determinant involved in oxidative and osmotic stress responses. In contrast to the PA14 katA mutant, the PAO1 katA mutant of was no less virulent as the corresponding wild type

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strain in both Drosophila and mouse models. Since PAO1 contains a forth catalase (KatM), which is expressed during the growth, we created the katAkatM double null mutant of PAO1 and found that it had little influence on its virulence. GeneChip analysis of transcriptome of both strains reveals that PAO1 strain has higher KatA level, which is concomitant with the higher expression of nitrate respiration genes in PAO1 than PA14. Ectopic expression of the fhp gene or inactivation of the nirS gene in the wild type PAO1 resulted in decreased KatA expression, suggesting the elevated KatA expression is attributed to NO accumulation. Based on these results, we suggest that the P. aeruginosa strains display differential NO accumulation and the subsequent differential KatA expression, which are associated with their differential virulence/survival mechanisms.


Quorum-quenching activity of an AHL-acylase from Pseudomonas putida KT2440Gudrun Koch1, Pol Nadal Jimenez1, Evelina Papaioannou1, Robbert Cool1 and Wim Quax1

1University of Groningen, The Netherlands

Although they are unicellular, bacteria are capable of cell-to-cell communication known as quorum sensing via small diffusible signalling molecules. N-acyl homoserine lactones (AHLs), the major communication molecules in Gram-negative bacteria have been shown to be responsible for the activation of many virulence genes in response to cell density. Pseudomonas putida strains can be found in the rhizosphere of plants where they exert plant-growth promoting actions and antagonistic behaviour against several plant pathogens. Up till now no AHLs have been detected in P. putida KT2440, neither any genetic evidence demonstrating the presence of any characterized quorum sensing system. Still, there is an indication that KT2440 has the ability to sense population density via a still unknown quorum-sensing system allowing successful interactions with the plant (1).Here we report the identification and characterization of a novel AHL-degrading enzyme in P. putida KT2440. PP_2901 was cloned and expressed in E. coli. PPu_2901 is, like its P. aeruginosa homologue, synthesized as an inactive proenzyme. Autocatalytic cleavage results in the active enzyme consisting of an α- and a β-subunit suggesting that is a member of the N-terminal nucleophile hydrolase superfamily. The enzyme shares a 54% amino acid identity to PvdQ from Pseudomonas aeruginosa PAO1 with the active site conserved. Like PvdQ, PP_2901 is able to hydrolyse C12-HSL (2). This research indicates that P. putida may use quorum quenching as an effective tool to interfere with plant pathogenic bacteria making it an effective biological control agent.(1) Espinosa-Urgel M, Ramos JL (2004). Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440. Appl Environ Microbiol. 70, 5190-8.(2) Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Camara M, Williams P, Quax WJ (2006). Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1. Infect Immun. 74, 1673-1682.


Pseudomonas aeruginosa PAO1 expresses enzymes with phospholipase A and phospholipase B activity Filip Kovačić1, Agathe Mandrysch2, Susanne Wilhelm2, Frank Rosenau2 and Karl-Erich Jaeger2

1Heinrich-Heine-University Duesseldorf, Institute for Molecular Enzyme Technology in Research Centre Juelich, 2Heinrich-Heine-University Duesseldorf, Institute for Molecular Enzyme Technology in Research Centre Juelich, IMET, Germany

Recently, it became increasingly obvious that phospholipases A and B (PLA and PLB) produced by several pathogenic Gram-negative bacteria play an important role during bacterial colonisation, invasion and pathogenesis [1]. Secreted and membrane-associated PLA and PLB activities were reported for the human pathogens Yersinia enterocolitica, Serratia liquefaciens, Helicobacter pylori, Legionella pneumophila, Streptococcus spp. and Plasmodium spp. Until now, the existence of phospholipase A or B activities in P. aeruginosa PAO1 was not reported.We have demonstrated for the first time that P. aeruginosa PAO1 produces both PLA and PLB activity. These activities are mostly cell-associated being present in membrane, periplasmic and cytosolic fractions; additionally, a low level of activity was detected extracellularly. The PLA and PLB activities are not affected by phosphate limitation or addition of choline and ethanol to the growth medium as observed for phospholipase C activity. We have analyzed the P. aeruginosa PAO1 genome for putative phospholipase encoding genes and have selected ten genes for further experimental examination. The systematic expression and purification of all candidate genes in P. aeruginosa and Escherichia coli resulted in the identification of four novel phospholipases which were named TesA, PlbF, PlaK and PlaB. TesA is a periplasmic protein possessing PLA, PLB and esterase activities. PlbF showed PLA, PLB, acyl-CoA thioesterase and esterase activities and is associated with the bacterial inner membrane. PlaK was localised in the cytoplasm and exhibited PLA and esterase activities. PlaB is a phospholipase A which is probably membrane-bound. Thus, we have identified a novel group of putative virulence factors produced by P. aeruginosa. P. aeruginosa plaK, plaB and plbF deletion mutants are currently tested to assess the contribution of these phospholipases for P. aeruginosa pathogenicity.[1] Taghrid S. Istivan and Peter J. Coloe (2006) Phospholipase A in Gram-negative bacteria and its role in pathogenesis, Microbiology 152:1263-1274.


MexT influences virulence determinants via a MexEF-OprN-independent pathway.Micheál Mac Aogáin1, Emilie Fargier1, Zhexian Tian1, Hazel O'Conner1, Marlies Mooij1, Claire Adams1 and Fergal O'Gara1

1BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland.

MexT, a LysR-type transcriptional regulator, positively regulates expression of the MexEF-OprN tripartite multidrug efflux system of Pseudomonas aeruginosa. MexT is expressed under routine laboratory conditions but is not thought to be active due to the absence of its natural co-inducer, hence mexEF-oprN is silent. However, mexEF-oprN is constitutively expressed in antibiotic resistant strains which carry mutation in mexS; a gene believed to influence the level of the unknown MexT co-inducer. In addition to antibiotic resistance, induction of mexEF-oprN leads to attenuation of in vivo virulence, reduced expression of homoserine lactone-dependent virulence traits including pyocyanin, elastase, rhamnolipids and the Pseudomonas Quinolone Signal (PQS) as well as reduced expression of the type three secretion system (TTSS). It is believed that MexEF-OprN mediates these effects via efflux of cell-signalling intermediates, which ultimately commits the cell to a state of reduced virulence.

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Here we explore the hypothesis that MexEF-OprN is not solely responsible for the phenotypic traits associated with MexT activation. We demonstrate that MexT can cause down-regulation of the TTSS and pyocyanin production in the absence of a functional MexEF-OprN efflux system. Hence we propose that MexT not only acts locally on mexEF-oprN, but also globally on diverse targets which constitute a novel regulon.The online MEME (Multiple Em for Motif Elicitation) tool was employed to identify a conserved motif in the upstream regions of genes which were co-expressed with mexEF-oprN in multiple transcriptome datasets. MexT over-expression lead to transcriptional activation of these targets in P. aeruginosa and Escherichia coli. Direct binding of MexT to the identified MexT target sequence was demonstrated by Electrophoretic Mobility Shift Assay (EMSA) and was abolished by the introduction of site specific mutation in the target sequence.


Membrane proteome analysis of Pseudomonas aeruginosa cultured in biofilm conditionsZofia Magnowska1, Sabrina Thoma2, Max Schobert2, Heinrich Lünsdorf3, Jürgen Wehland3 and Lothar Jänsch3

1Helmholtz Centre for Infection Research , 2Technical University, Biozentrum, Braunschweig, Germany, 3Helmholtz Centre for Infection Research, Braunschweig, Germany

Pseudomonas aeruginosa (PA) colonize the lung of cystic fibrosis (CF) patients and finally develop a chronic infection which causes tissue damage and consequently the death of patients. An efficient strategy for vaccination or at least sensitive diagnostics of the infection state is necessary to interfere with this process. However, only few Pseudomonas aeruginosa (PA) antigens were validated so far, and they do not serve effectively as diagnostic or prognostic markers or vaccine candidates. Finding novel antigenic candidates is the object of this study.Due to the fact that the membrane forms the first line of contact for the host's immune system we decided to search the membrane proteome comprehensively for new potential antigens. LC-MS was used to investigate both associated and transmembrane proteins representatively, whereas the enrichment of pure membranes itself was accomplished by using isopycnic gradient centrifugation. Importantly this approach can discriminate between membrane fractions containing inner and outer membranes and outer membrane vesicles as observed by electron microscopy. Ongoing work focusses on the detailed biochemical and functional analysis of these vesicles that might play a significant role in adaptive immune response as well as quorum sensing.To improve the chance for the detection of antigens actually playing a significant role in CF, bacteria are cultivated under a condition mimicking real infection situation: PA biofilm was established on nitrocellulose filters, which allow for extended growth and the cultivation was performed in artificial sputum medium. This medium is an appropriate model of chronic lung colonization and is useful in achieving CF-specific phenotypes of PA. Furthermore, the three-dimensional structure of lung biofilm establishes structural and functional compartmentalisation. In some of the regions oxygen saturation is strongly reduced in particular in the late state of infection. In this study, regulation of protein expression in the membrane fraction under aerobic or anaerobic conditions that are characteristic for early and late states of infection were comparatively analysed with quantitative LC-MS/MS. In conclusion, we have achieved excellent access to the membrane proteome getting insight into the physiological adaptations that depend on the presence of oxygen.


Gene expression characteristics of a virulent early CF isolate of Pseudomonas aeruginosa grown in artificial sputum mediumJim Manos1, Carina Fung2, Cynthia Whitchurch3, Lynne Turnbull3, Torsten Seeman4, Torsten Thomas5, Jonathan Arthur2, David Armstrong6, Barbara Rose2 and Colin Harbour2

1University of Sydney, 2University of Sydney, Sydney, Australia, 3University of Technology, Sydney, Australia, 4Victorian Bioinformatics Consortium, Monash University, Melbourne, Australia, 5University of New South Wales, Sydney, Australia, 6Monash Medical Centre, Melbourne, Australia

The emergence of virulent Pseudomonas aeruginosa clones is a major new threat to the health of patients with cystic fibrosis (CF) in several continents. Frequent clones such as the Australian Epidemic Strain-1 (AES-1) result in more hospitalisations and greater exacerbations in CF patients. Global gene analysis of CF strains has been hampered by the lack of an array template containing CF-strain specific genes and by lack of appropriate media mimicking CF lung sputum. In order to investigate global gene expression in virulent strains, we sequenced the genome of an AES-1 clonal isolate (AES-1R) taken from a child with CF and associated with a fatal Melbourne outbreak of virulent P. aeruginosa. We subsequently used the gene sequence together with that of seven other sequenced P. aeruginosa isolates, both clinical and environmental, to design a non-redundant array (PANarray) for detection of strain-specific genes of interest. P. aeruginosa AES-1R and PAO1 were grown in an Artificial Sputum Medium (ASMDM) to mimic CF lung sputum, and gene expression compared with cells grown in Luria-Broth culture using the PANarray. Results from biological replicates showed AES-1R differentially expressed 75 genes (p<0.01 fold-change > 2×) while PAO1 did not differentially express any genes with fold-change > 1.5×. Interestingly, 56 differentially expressed genes (75%) were AES-1 specific, three were shared with the Manchester clonal CF strain (MA) and only two were shared with the wound isolate PAO1, demonstrating the importance of the strain-specific array and the artificial sputum medium. Upregulated genes with known functions included ferric enterobactin receptor pfeA involved in heterologous iron uptake (+9.9×), phosphotyrosine protein phosphatase ptpA (+3.9×) and a putative cyanate MFS transporter (+4.2×), while downregulated genes included arginine deaminase (-3.6×) and the transcriptional regulator MvaT (-2.4×). Iron acquisition by the high affinity siderophore enterobactin suggests enhanced growth by AES-1R in ASMDM is iron-mediated. Identification of virulence genes should lead to more rational treatment strategies for CF patients infected with virulent strains of P. aeruginosa.


Control of persister frequency in Pseudomonas aeruginosa in response to environmental factorsNina Moeker1, Charles R. Dean1, E. Peter Greenberg2 and Jianshi Tao1

1Novartis Institutes for BioMedical Research, 2University of Washington, USA

Pseudomonas aeruginosa is an important opportunistic pathogen that causes a variety of infections. Similar to other bacteria, Pseudomonas aeruginosa produces a small fraction of seemingly dormant, multidrug-tolerant cells known as persisters, which are not eradicated during antibiotic exposure (1). Given the increasing problem of intractable infections caused by P. aeruginosa, and the current incomplete understanding of the contribution of persisters, we undertook to examine persister formation in P. aeruginosa. We demonstrated that even though P. aeruginosa produces less persisters (up to 0.01%) than does Escherichia coli (up to 1%) or Staphylococcus aureus (up to 10%), it has the unique ability to increase the persister cell fraction up to 100fold in response to exogenous pyocyanin (PYO) or paraquat. Because both compounds are redox-active, we tested for persisters under anaerobic conditions.

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Intriguingly, we found that PYO added to anaerobically grown P. aeruginosa cultures rendered close to 100% protection against killing by certain antibiotics. To extend these observations, we tested for increased P. aeruginosa persister numbers in response to several conditions. Osmotic stress, heat shock, or treatment with ofloxacin had no effect, whereas spent growth media from the strains PA14, PAO1, and S. aureus, tetracycline, and human serum significantly increased persister numbers in logarithmic phase cultures. This increase allowed us to isolate enough P. aeruginosa PA14 persisters for total RNA isolation and transcriptional profiling to gain insight into the global transcriptional changes underlying persistence in P. aeruginosa. Preliminary analysis indicates the repression of genes involved in energy metabolism or catabolic processes and the activation of genes coding for heat shock proteins, consistent with the overall scenario reported for E. coli and suggestive of metabolic dormancy (2). We are currently focused on characterizing individual transcriptional changes that seem to be unique for P. aeruginosa.(1) K. Lewis. 2007. Nat Rev Microbiol. 1: 48-56(2) D. Shah et. al. 2006. BMC Microbiol. 6:53.


Molecular detection of the entomopathogenic bacterium Pseudomonas entomophila using polymerase chain reaction (PCR)Dimitris Mossialos1, Antonios Papagiannoulis1, Konstantinos Mathiopoulos1 and Panagiotis Markoulatos1

1University of Thessaly, Department of Biochemistry & Biotechnology, University of Thessally, GR-41221 Larissa, Greece

Aims: To develop a specific, fast and simple molecular method useful to detect the entomopathogenic bacterium Pseudomonas entomophila.Methods and Results: The use of bioinformatics tools (available in TIGR) allowed us to identify unique genes present in P. entomophila genome but not in other published pseudomonad genomes. Using the DNA sequence of such genes, we designed diverse primers aiming to detect specifically P. entomophila by PCR. Moreover we used an already published pair of primers designed to amplify 16S rDNA in Pseudomonas species (Spiker et al. J. Clin. Microbiol. Vol. 42: 2074-2079). Using chromosomal DNA extracted from diverse environmental pseudomonad and non-pseudomonad species we performed PCR reactions under variable experimental conditions, checking the specificity of our primers as well as the published 16S rDNA primers. A 618 bp fragment was amplified only in Pseudomonas, including P. entomophila, using the 16S rDNA primers. Primers designed to amplify gene PSEEN 1497, amplified specifically a 570 bp fragment in P. entomophila. Furthermore we developed a duplex PCR, combining 16S rDNA and PSEEN 1497 primers, which allowed us to detect P. entomophila present in experimentally infected Drosophila melanogaster.Conclusions: We developed a specific, fast and simple duplex PCR useful for P. entomophila detection, present in bacterial cultures or directly from infected insects.Significance of the Study: To our best knowledge this is the first molecular method aiming to detect P. entomophila in environmental samples (bacterial isolates or insects). The use of our method will facilitate studies related to ecology and insect host range of this entomopathogenic bacterium.


Monitoring sputum populations of the Pseudomonas aeruginosa Liverpool epidemic strain in cystic fibrosis patients during pulmonary exacerbations reveals significant phenotypic and genotypic diversificEilidh Mowat1, Joanne L. Fothergill1, Helen Miller2, Martin J. Walshaw2, Martin J. Ledson2 and Craig Winstanley1

1Division of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Liverpool, UK, 2Regional Cystic Fibrosis Unit, Cardiothoracic Centre, Liverpool, UK

Recent studies have revealed that several transmissible epidemic strains of Pseudomonas aeruginosa are widespread throughout CF clinics across the UK and elsewhere. The most predominant clone is the Liverpool epidemic strain (LES), which is associated with greater patient morbidity in the UK. The genome sequence of LESB58 has enabled identification of several novel mobile regions implicated in the pathogenicity and success of this epidemic strain.Sputum samples from CF patients were taken during periods of stability and sequentially throughout exacerbations (at the initial presentation of exacerbation, three days into intravenous antibiotic therapy and at the end of treatment). From each respective sample 40 LES isolates were analysed for population fluctuations using a series of high-throughput phenotypic and genotypic assays.During the sampling period variations in colony morphology, auxotrophy, hypermutability, pyocyanin/LasA production and antibiotic susceptibility profiles were observed in LES populations. PCR screening for mobile genomic islands and LES-specific prophages revealed significant diversification in the presence or absence of these novel regions. PCR examination of filter-sterilised, DNase-treated, sputum samples enabled the identification of LES-specific prophages directly from patient sputa.This study indicates that LES populations of P. aeruginosa fluctuate and adapt to antibiotic treatment during pulmonary exacerbations. The adaptability of this strain may enhance its ability to transmit and contribute to the overall virulence of this epidemic pathogen within CF patients.This work was funded by the Dr Hadwen Trust and the NIHR as part of the Biomedical Research Centre in Microbial Diseases.


PvdQ: an acylase with multiple functions in Pseudomonas aeruginosaPol Nadal Jimenez1, Gudrun Koch1, Evelina Papaioannou1, Robbert Cool1 and Wim Quax1


The successful survival of Pseudomonas aeruginosa lies in its incredible ability to adapt to various environments. In order to create an advantage over other co-inhabitants in a bacterial niche, P. aeruginosa has developed strategies to change rapidly from host-invasive mode to survival mode. Secreted diffusible signalling molecules are key components of inter cell communication and in many cases they trigger a virulence response. P. aeruginosa produces three major autoinducer molecules, C4-homoserine lactone (C4-HSL), 3-oxo-C12 homoserine lactone (3-oxo-C12-HSL) and the Pseudomonas quinolone signal (PQS), whose productions are tightly interrelated. Production of 3-oxo-C12-homoserine lactone induces transcription of pyocyanin, elastase and several other virulence factor genes. If the

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conditions in the niche turn less favourable either by a decrease in available nutrient sources or by an increase in competing co-inhabitants, this bacterium reduces the production of virulence factors and starts scavenging. Iron has been proven to be one of the primary limiting factors for many organisms. In the present study we provide evidence indicating that under iron limiting conditions, expression of pvdQ not only lowers the production of virulence factors via degradation of 3-oxo-C12-HSL but also significantly increases biofilm production and swarming motility [1]. All together this data suggests that the role of PvdQ in P. aeruginosa may go far beyond quenching 3-oxo-C12-HSL [2].[1] Overhage J, Bains M, Brazas MD, Hancock RE. (2008). Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol. 190 (8):2671-9.[2] Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Camara M, Williams P, Quax WJ (2006). Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1. Infect Immun. 74, 1673-1682.


Proteomic analysis of Pseudomonas aeruginosa strains in response to bronchial epitheliaJulie Anne O'Callaghan1, Heidi Mulcahy1, Christine Baysse1, Claire Adams1 and Fergal O'Gara1

1BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland

During infection Pseudomonas aeruginosa exerts tight regulatory control over gene expression to counteract host defense mechanisms and facilitate the establishment of infection. The post-transcriptional regulator RsmA plays a key role in controlling a number of traits associated with pathogenicity (Goodman et al., 2004; Burrowes et al., 2005). We demonstrated that RsmA mediates regulation of the type III secretion system (T3SS) with subsequent effects on invasion of and cytotoxicity towards epithelial cells in an in vitro infection model (Mulcahy et al., 2006). RsmA is also involved in colonization and dissemination in vivo in a mouse model of acute pneumonia (Mulcahy et al., 2008).A proteomics approach was employed to identify P. aeruginosa proteins that play a role during infection and to characterize the global effects of RsmA during the initial interaction of P. aeruginosa with host tissues. Total protein profiles were generated from PAO1 and an rsmA mutant, after 1.5 h interaction with human airway epithelial cells in order to specifically identify proteins with significantly altered expression during infection of bronchial epithelial cells. Predictably, proteins differentially regulated in response to human airway epithelial cells included those involved in stress response and energy conservation. However, ongoing work has focused on an uncharacterized sensor protein induced during host cell interactions and which appears to be regulated by RsmA. Inactivation of the sensor protein led to increased invasion of but reduced cytotoxicity towards epithelial cells compared to its wild type, confirming that it plays a role during infection. Further characterization of the sensor suggests a role in modulating the expression of the T3SS in response to host cell infection. Uncharacterized homologues of the sensor also exist in other pathogenic bacteria presenting the possibility of interspecies significance. Therefore, this sensor may play a key role in microbial host interactions and may be a potential target for the development of novel antimicrobial therapy.Mulcahy H. et al. Infect. Immun. 2006;74:3012-5. Mulcahy H. et al. Infect. Immun. 2008;76:632-8.Burrowes E. et al. Micro. 2006;152:405-18.Goodman J.L. et al. Dev. Cell. 2004;745-54.


The quorum quenching acylase PvdQ reduces the pathogenicity of P. aeruginosa in Caenorhabditis elegansEvelina Papaioannou1, Mariana Wahjudi1, Pol Nadal Jimenez1, Gudrun Koch1 and Wim Quax1


Pseudomonas aeruginosa PAO1 gene PA2385 encodes an acyl-homoserine lacton (AHL) acylase (PvdQ) capable of degrading 3-oxo-C12-HSL by cleaving the AHL amide and thus releasing the fatty acid chain from the homoserine lactone nucleus. PvdQ has been proven to function as a quorum quencher in vitro, by a number of phenotypic assays [1]. To address the question whether PvdQ can show quorum quenching properties also in vivo, the nematode C. elegans was used as an infection model; a simple model host that can be infected and killed by a remarkably large number of human pathogens [2,3]. The ability of PvdQ to act as a quorum quencher in the C. elegans model was investigated under different conditions by simply replacing the normal laboratory food source of the nematodes, Eschericia coli OP50 (E. coli OP50), with our Pseudomonas strains (PAO1 WT, PAO1-pME-PvdQ, PAO1-ΔPvdQ, PAO1-ΔlasRlasI) and monitoring the survival of the nematodes throughout a 4 day course. The results of both the fast and slow killing assays revealed a reduction in the pathogenicity of P. aeruginosa when PvdQ is overexpressed. Under a standard paralysis assay more than 75% of the nematodes exposed to PAO1(pMEPvdQ) survived and continued to grow using this strain as a food source. Addition of purified PvdQ protein to PAO1 resulted in reduced pathogenicity and increased the lifespan of the nematodes compared to the wild type. From our observations we can conclude that PvdQ might be a strong candidate for anti-bacterial therapy against Pseudomonas infections. [1] Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Camara M, Williams P, Quax WJ (2006). Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1. Infect Immun. 74, 1673-1682.[2] Larry A. Gallagher and Colin Manoil (2001) Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by Cyanide Poisoning. J. Bacteriol 183(21): 6207-6214 [3] Man-Wah Tan and Frederick M Ausubel (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Current Opinion in Microbiology 3 (1): 29-34


Ptsp gene involved in elastin hydrolysis in Pseudomonas aeruginosaMingqiang Qiao1, Yongxin Jin2, Hongming Yao2, Liwei Liu2, Xiaoyuan Mi2, Rui Mou2, Quanmin Liu2, Xiuming Zhang2, Yanling Bai2 and Haijin Xu2

1College of Life Science, NanKai University, 2The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, China

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen responsible for a wide range of acute and chronic infection. When growing in host, it secretes a large number of toxic and degradative enzymes, including several proteolytic enzymes

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which play important roles in pathogenesis. The most abundant secreted protease of P. aeruginosa is elastase (encoded by the lasB gene), which is considered as a major virulence factor.In this work, artificial Mu transposome technology was utilized to explore the candidate genes involved in the hydrolyzing ability towards elastin of P. aeruginosa. Eight mutants with altered levels of elastase production were isolated by investigating the hydrolyzing circles of the strains on the elastin agar and the elastin-congo-red assay. Among all mutants, B84 (ptsP::Mu) has the most remarkable phenotypic change, whose elastase activity is 13% of that in the wild type and the lasB promoter’s activity is just 7% of that in the wild type. However, it is intriguing that the lasI (a gene of quorum sensing system, positively regulating lasB) promoter’s activity increases to 192% in B84. These results presented a connection between gene ptsp and elastase production and showed the complexity of the quorum sensing regulation.Supported by the National Natural Science Foundation of China (30570089), the Plan of Application and Foundation Research of Tianjin(07JCYBJC08000) and the Developing Plan of Science and Technology of Tianjin (06YFGZSH07100)


Pseudomonas aeruginosa population biology in COPDElza Rakhimova1, Lutz Wiehlmann1, Aimee L Brauer2, Sanjay Sethi2, Timothy F Murphy2 and Burkhard Tümmler1

1Medizinische Hochschule Hannover, Germany, 2State University of New York at Buffalo, USA

Airways infection with Pseudomonas aeruginosa is common in individuals with bronchiectasis or cystic fibrosis (CF). It is a common and important cause of hospital acquired infection, particularly in patients in intensive care units and those undergoing mechanical ventilation. P. aeruginosa is also isolated from the sputum of adults with chronic obstructive pulmonary disease (COPD), but the role of the organism in the course of the disease is less well characterized than in other clinical settings.The goal of the present study is to elucidate the population structure of sequential P. aeruginosa isolates collected in a 10-year prospective study from 39 of 126 adults with COPD acquired P. aeruginosa and to compare the genotypes of the COPD airways isolates with those of isolates from CF patients with chronic P. aeruginosa infection.The typing of 134 P. aeruginosa COPD isolates with a microarray uncovered 60 unrelated bacterial clones. The worldwide dominant clones PA14 and clone C were the most abundant clones among the COPD isolates.The carriage patterns of P. aeruginosa were different in CF and COPD. Thus, the intraclonal microevolution and a frequent turnover or loss of clones are typical for the infections with P. aeruginosa in COPD. Only seven of the thirty-nine patients (18%) were persistently colonized with the same P. aeruginosa clone for several years. In contrast, 60% of the chronically colonized CF patients at the CF clinic Hannover were still harbouring the initially acquired P. aeruginosa clone after more than ten years of colonization.


Isolation and characterization of small colony variants of Pseudomonas aeruginosa grown under simulated urinary tract conditionsPetra Tielen1, Katja Heinemann1, Marie Sauer2, Katrin Zapf2, Rainer Krull2 and Dieter Jahn1

1Technische Universität Braunschweig, Institute of Microbiology, Spielmannstr. 7, D-38106 Braunschweig, Germany, 2Technische Universität Braunschweig, Institute of Biochemical Engineering, Gaußstr. 17, D-38106 Braunschweig, Germany

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe acute and chronic infections, especially in immunocompromised patients. Common infections caused by P. aeruginosa are complicated urinary tract infections, mostly in association with indwelling catheters, where it is causative agent in 35 per cent of the cases. Due to its high intrinsic antibiotic resistance and its diverse adaptation strategies such infections often become persistent.We developed a biofilm reactor mimicking urethral catheter infections using an artificial urine medium (AUM) with a continuous flow rate of 1.4 l per day. P. aeruginosa isolates from acute urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs) were incubated under these conditions in comparison to the lab strain PA14. After an incubation time of seven days we observed small colony variants (SCVs) in a concentration of up to 10 % of the whole population. A comprehensive physiological characterization of the obtained SCVs showed global changes in phenotypic patterns in relation to their parent strains, e. g. increased antibiotic resistances, increased biofilm formation and decreased motilities. Moreover, several SCVs showed an enhanced cell aggregation.Interestingly, SCVs were also isolated from chronic CAUTIs, which indicate that phenotypic variations to SCVs might be a common strategy of P. aeruginosa to cause persistent infections in the urinary tact.


Bistable virulence gene expression in Pseudomonas aeruginosa requires positive feedback of a LysR-type transcription activatorKeith H. Turner1, Isabelle Vallet-Gely2 and Simon L. Dove1

1Children's Hospital Boston - Harvard University, USA, 2CNRS-CGM, Gif sur Yvette, France

Bistable gene expression can generate phenotypic diversity in clonal populations of bacteria. Here we uncover a bistable switch in the opportunistic human pathogen Pseudomonas aeruginosa. This switch controls the expression of a small subset of genes including aprA, which encodes the virulence factor alkaline protease. We present evidence that bistable expression of regB (regulator of bistability), which encodes a LysR-type transcription activator, mediates this switch. In particular, using DNA microarrays, quantitative RT-PCR analysis, chromatin immunoprecipitation and reporter gene fusions we identify genes directly under the control of regB and show that these genes are bistably expressed. Furthermore, we present evidence that regB is itself bistably expressed and that its expression is positively autoregulated. Finally, using single-cell analysis of a GFP reporter fusion, we present evidence that this positive autoregulation of regB is necessary for bimodal expression of the regulon. Our findings reveal a previously undescribed bistable switch that controls virulence gene expression in P. aeruginosa, and suggest that it may be mediated, at least in part, by positive feedback of a LysR-type transcription activator.

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Topic: Pseudomonads as Pathogens

P. syringae efflux pumps. Fighting on multiple fronts.Paola Andrea Vargas Gallego1, Antonia Felipe1 and Mari-Trini Gallegos1

1Estación Experimental del Zaidín (CSIC), Granada, Spain

Plants elaborate a vast array of natural products, many of which have evolved to confer selective advantage against microbial attack. Flavonoids, isoprenoids, and alkaloids compose three major classes of secondary metabolites synthesized by higher plants that have been implicated in a broad range of physiological processes and considered to be important players in the protection of plants against potential pathogens. However, bacteria resistant to the action of these compounds have developed protective mechanisms. The most effective and widespread method of resistance is the extrusion of toxic compounds by multidrug transporters in an energy-dependent process. So far, only a limited number of transporters conferring multidrug resistance in phytopathogenic bacteria have been characterized and their protective role established. In particular, knowledge regarding existence and the role of these transporters in Pseudomonas syringae is limited; nevertheless some data has recently been reported. The role of the MexAB-OprM efflux pump in the infection process of plants by P. syringae pv. tomato DC3000 has been demonstrated since a ΔmexAB-oprM mutant is impaired in its ability to multiply in the plant and cause symptoms. Also, the absence of the MexAB-OprM pump increases the sensitivity of the mutant strain to a large number of antimicrobial compounds compared with the wild type strain (Stoitsova et al., 2008). The aim of this study was the physiological and molecular characterization of the the RND-type transporter MexAB-OprM of P. syringae pv. tomato DC3000. Located 5' with respect to the mexAB-oprM structural genes is PSPTO_4302 ORF coding for a regulator (pmeR) that belongs to the TetR family of transcriptional regulators. The strain ΔpmeR (mutant deficient in the regulator PmeR) exhibited MIC values higher than those of the wild strain for most of the compounds tested. We also assayed the expression of the PmexA and PpmeR promoters in the wild type and ΔpmeR mutant and found higher levels of gene expression in the ΔpmeR strain. These results indicate that this regulator is an auto-repressor that also controls the expression of mexAB-oprM operon in response to different antibiotics and plant secondary metabolites.


Development of bioluminescent Pseudomonas aeruginosa marker strains for infection monitoring in vivoFlorian Woelbeling1, Antje Munder2, Burkhard Tuemmler1,2 and Ulrich Baumann2

1Hannover Medical School, Clinical Research Group, 2Department of Pediatric Pulmonology and Neonatology, Germany

Classic infection models in rodents innoculate the microbe of interest and then recover the pathogen at a certain time-point from selected organs. The in vivo imaging system (IVIS) developed by Xenogen allows continuous monitoring of the course of infection by non-invasive imaging of recombinant bioluminescent bacteria. Our interest refers to strategies that prevent chronic Pseudomonas aeruginosa infection in Cystic Fibrosis patients. Vaccination would be a sensible approach. In order to better represent the course at the onset of the P. aeruginosa airway infection in vivo, we are currently establishing a model of low dose nasal inocculation and longitudinal investigation of physiological and spirometrical parameters, combined with the investigation of the internal localization of the bacteria by using bioluminescently labelled Pseudomonads. Several strains of P. aeruginosa, including the well-characterized and clinical relevant strain PAO1 and TB, were bioluminescently labelled by stable integration of a mini-CTX-lux transposon (12538 bp), which is inserted at the attB site of the Pseudomonas genome. Luminescence is measured using the Xenogen IVIS 200 system, which measures emitted photons using a highly-sensitive CCD camera. In vitro experiments of the labelled strains resulted in the very high values of bioluminescence of about 109 photons/sec/cm2/sr. A laser scanner provides 3D surface topography for the localization of internal sources of luminescence. This non-invasive method can be used to monitor the localization and quantity of Pseudomonads in mice in vivo. Initial in vivo experiments showed that a lung infection with bioluminescent P. aeruginosa can be traced up to 24 - 48 hours post infection. Further experiments will be directed to investigate whether vaccination with a recombinant P. aeruginosa outer membrane fusion protein (OprF-OprI) can prevent infection in C57BL/6 wildtype and also CFTR-deficient mice.


Characterisation of Cystic Fibrosis isolate Pseudomonas aeruginosa 18A biofilm phenotypes and morphotypic variantsJerry Khee Keong Woo1, Janosch Klebensberger2, Scott Rice2, Sylvia Kirov3 and Staffan Kjelleberg2

1University of New South Wales School of Biotechnology and Bio-molecular Sciences, 2School of Biotechnology and Biomolecular Sciences, Centre for Marine Bio-Innovation, University of New South Wales, Sydney, NSW 2052, Australia, 3School of medicine, University of Tasmania Clinical School, 43 Collins St, Hobart, TAS7001, Australia

Pseudomonas aeruginosa is a Gram negative opportunistic pathogen which is a primary cause of mortality in Cystic Fibrosis (CF) patients. Previous studies have suggested that P. aeruginosa exists within the patients' lungs in the form of biofilms. One feature of chronic infections, especially in CF isolates, is the formation of morphotypic variants, such as small colony variants (SCVs). The appearance of such variants appears to be a key step in the establishment of chronic infection strains and therefore it is important to understand the factors that are responsible for the generation of genetic variants. We have recently shown that P. aeruginosa 18A generates extensive morphotypic diversity during in vitro biofilm development and compared this phenomenon across clinical CF isolates (Kirov et al. 2007, Microbiology, 3264-3274). Using P. aeruginosa 18A, a clinical CF isolate, we now report that this strain shows greater colony morphotypic diversity (including small colony variants, large colony variants and small colonies with translucent edge and yellow centre) than PAO1 (small colony variants) and that 18A has a 5-fold higher mutation rate. In addition to morphotypic variation, we observed functional variation within isolates of the same morphotype (e.g. using Biolog GN2 plates for differences in carbon source utilisation), as well as changes in total protease, elastase, quorum sensing and motility (swimming, swarming and twitching). Moreover, the SCVs of 18A biofilm dispersal variants generally showed greater heterogeneity in elastase, protease and quorum sensing than was observed in the mucoid isolates. We conclude that in vitro biofilms generate genetic variants that have phenotypes similar to those observed for clinical isolates in situ.

Cell Surface, Transport and Secretion

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Oral presentations

O-17 Topic: Cell Surface, Transport and Secretion

Assembly of type IV pili in Pseudomonas aeruginosaLori L. Burrows1

1McMaster University, Hamilton, Canada

Type IV pili (T4P) are used by many species, including Pseudomonas, for attachment to surfaces, DNA uptake, bacteriophage adsorption and surface-associated twitching motility. T4P are important virulence factors that mediate interactions with host epithelia and are involved in initiating and modulating biofilm structure. Our investigations of P. aeruginosa T4P focus on defining the specific functions of the large number of gene products implicated in pilus assembly, as most mutants have a similar and uninformative non-piliated phenotype. We want to understand how T4P assemble through the complex Gram-negative cell envelope in a manner that allows them to both generate and withstand strong retraction forces during the process of twitching motility. Using bioinformatic analyses of the available P. aeruginosa genomes, we have determined which components of the system are highly conserved and which are variable, helping to clarify how the observed sequence variations in the surface-exposed major pilin subunit protein can be accommodated by the assembly system. Using single and double mutant analyses, we have distinguished between proteins that are essential for assembly versus those that involved in, but dispensable for, assembly. The latter components are likely to control assembly rates by modulating protein-protein interactions. Our mutant phenotypic analyses have also demonstrated a more extensive functional integration of the T4P system with the structurally related Xcp type II secretion system responsible for secretion of various protein toxins and enzymes than was previously appreciated. Biochemical and structural biology efforts aimed at conserved components of the assembly system are beginning to reveal new insights into its architecture. Our studies of the PilMNOPQF and FimV proteins reveal patterns of mutual stabilization, localization and functional association, suggesting that they form inner and outer membrane protein complexes involved in pilus assembly. These proteins are widely conserved among T4P producing bacteria and likely represent a core complex for T4P assembly.


The "P-usher", a novel protein transporter involved in fimbrial assembly and TpsA secretionSégolène Ruer1, Geneviève Ball2, Alain Filloux3 and Sophie de Bentzmann2

1CNRS , 2CNRS LISM UPR9027 31, Chemin Joseph Aiguier 13402 Marseille France, 3Centre for Molecular Microbiology and Infection Division of Cell and Molecular Biology Faculty of Natural Science Flowers Building, 1st floor South Kensington Campus Imperial College London London SW7 2AZ, UK

Bacterial pathogenesis can be tightly related to protein secretion and assembly of cell surface appendages. Type Vb secretion system involves two partners (TPS), called TpsA and TpsB. TpsA protein is secreted at the surface and is frequently an adhesin, such as the filamentous hemagglutinin (FHA) from Bordetella pertussis. The TpsB protein is the outer membrane protein forming the beta-barrel pore through which the TpsA substrate is secreted. The TpsB protein possesses a domain called POTRA (polypeptide-transport-associated domain) that has been described to play a role in interaction, folding and TpsA secretion. Pseudomonas aeruginosa possesses 5 complete copies of TPS and one tpsA orphan gene and is equipped with 5 fimbrial structures assembled by the chaperone-usher pathway (Cup) in the genome of PAO1 strain. The CupB system contains a gene, cupB5, the tpsA orphan gene.We previously showed that in absence of the usher CupB3, CupB fimbriae are not assembled. Interestingly, the TpsA protein CupB5 secretion is dependent of the CupB3 usher. This CupB3 usher contains a unique POTRA-like domain that is not present in the other ushers. We revealed that this POTRA domain is not essential for the TpsA secretion, since CupB5 secretion still occurred in a POTRA-domain mutant of the cupB3 gene. In this mutant, CupB machinery is not able to assemble CupB fimbriae, assembly which was restored in a cupB5 mutant. Additionally, we showed that CupB5 sticks to CupB fimbriae at distance form the bacterial cell surface and probably uses CupB fimbriae to move away from the bacterial cell surface. We also showed a slight effect of CupB5 in biofilm formation. The CupB3 bi-functional protein, renamed the P-usher, is therefore a probable mosaic protein resulting from the fusion of a C-terminal beta-barrel domain of an usher and an N-terminal POTRA domain of another TpsB beta-barrel protein. This evolutionary hypothesis was confirmed when examining this cupB cluster in Pseudomonas fluorescens Pf5 (PF) genome which contains a cupB3 gene encoding a classical usher and a gene encoding a TpsB protein. We were able to express this tpsBPF gene in P. aeruginosa, in absence of its CupB3 usher. The P. aeruginosa CupB5 secretion occurred through the TpsB protein of P. fluorescens. The P. aeruginosa CupB fimbriae are new composite heteropolymeric fimbriae exposed at the fimbrial subunits and FHA-like protein which respective role in the interaction with the host remains to be elucidated.


Posttranslational regulation of type VI secretion in Pseudomonas aeruginosaJoseph David Mougous1

1University of Washington, Seattle, USA

The type VI secretion system (T6SS) is a widespread, recently described protein secretion pathway that can deliver its substrates directly into host cell cytoplasm. The genome of Pseudomonas aeruginosa PAO1 encodes three apparent T6SSs. One of these, encoded by Hcp Secretion Island I (HSI-I), is regulated in a complex fashion at both posttranscriptional and posttranslational levels. Posttranscriptional control of the HSI-I-encoded T6SS (H1-T6SS) is stringent and occurs via the Gac/Rsm pathway, with RsmA functioning as a translational repressor. Even with high levels of T6S proteins, activity of the system is minimal until it is triggered at the posttranslational level by a serine-threonine kinase, termed PpkA. Using a small molecule induced dimerization system, we show that PpkA becomes activated when dimerized, and that its dimerization is sufficient to trigger the secretion system. In addition, we provide evidence that autophosphorylation of the kinase ensues and is used to recruit the kinase substrate, Fha1. Using fluorescence microscopy and immunoprecipitation, we show that Fha1 is in complex with other H1-T6SS proteins, including ClpV1, prior to and following its recruitment to the activated kinase. Finally, we report the identification of several additional proteins that participate - both positively

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and negatively - in the posttranslational regulatory pathway of the H1-T6SS. One of these proteins, which we term TagR, may function as a PpkA co-receptor for the recognition of the H1-T6SS activation signal. When taken together with other recent findings in the field, we are able to present a structure-function model for the T6SS.


Biogenesis of the type III secretion system in P. aeruginosaIna Attrée1

1CNRS, CEA, University Joseph Fourier, Grenoble, France

Type III secretion (T3S) machineries are present in most Gram-negative bacteria and are devoted to the secretion and injection of bacterial toxic proteins directly into the target cell cytoplasm. The cytotoxicity of P. aeruginosa strains is dependent on active T3S system which is able to deliver four distinct effectors ExoS, ExoT, ExoY and ExoU, all of which play important roles in eukaryotic cell intoxication. The secretion apparatus is built up on the bacterial surface in regulated, sequential manner and is composed of about 20 proteins. The basal part of the injectisome is imbedded within the two bacterial membranes and is prolonged by an 80 nm-long needle-like structure which serves as a secretion channel. In P. aeruginosa, the needle is composed of a 7-kDa protein, PscF encoded by the exsDpscBCDEFGHIJKL operon. We showed that PscF has an intrinsic property to polymerise into long fibres of 8 nm in width and >1 micrometre in length. The polymerisation is controlled in bacterial cytoplasm by two chaperones PscE and PscG, which form a tight heterotrimeric complex with PscF. The 2-Å x-ray crystal structure of the complex revealed the unique mechanism of chaperon-substrate interaction: a TetratricoPeptideRepeat (TPR)-domain of PscG sequesters the amphipatic C-terminal helix of PscF through multiple interactions. Indeed, in vivo and in vitro, this helix was shown to be absolutely required for polymerisation of PscF and the function of the secretion apparatus. Mutations introduced within the TPR domain of PscG caused the loss of cytotoxicity in a virulent P. aeruginosa strain. PscE folds -helixes and interacts only with PscG. In order to assign aα in 3 function to PscE we constructed strains in which PscE was either lacking one of the helices or possessing mutations in amino acids predicted to interact with PscG. The screen of more than 20 amino acids revealed several hot spots of interaction. The phenotypic analysis of the mutants showed that the PscE protein plays an important role in the stabilisation of the complex, in particularly of PscG. Preliminary results indicate that overexpressed PscG and PscF are able to overcome the absence of PscE, and that the recognition and the export of PscF by the secretome requires only the PscG chaperon.


PO-185 Removed by request of the author before online publication.

PO-186 Topic: Cell Surface, Transport and Secretion

Alginate polymerisation/secretion in Pseudomonas aeruginosaBernd HA Rehm1

1Massey University, New Zealand

Pseudomonas aeruginosa causes severe chronic infections in the lungs of cystic fibrosis (CF) patients. Establishment of a chronic CF lung infection coincides with production of copious amounts of the exopolysaccharide, alginate, resulting in formation of persistent biofilms. Although the entire alginate biosynthesis gene cluster in P. aeruginosa was identified about two decades ago, the key enzyme, the alginate polymerase, has not been functionally assigned. The alginate polymerase has been proposed as a multiprotein complex composed of the subunits Alg8, Alg44, AlgX and AlgK [1]. AlgK, AlgX and Alg44 were found to be predominantly localized in the periplasm. Currently, only the protein-protein interaction between AlgX and MucD (serine protease) was experimentally shown [2] and promoter activity studies suggested a new link between polymerisation and gene regulation which will be discussed in this presentation. Recently, the protein Alg8, which resembles glycosyltransferases, was identified as key membrane protein for the production of alginate [3, 4]. Here, the refined membrane topology of Alg8 as well as a proposed reaction mechanism based on site-specific mutagenesis will be discussed. Alg44, which is essential for alginate production, was found to bind the activator c-di-GMP required for alginate biosynthesis [5]. In this study, MucR (PA1727) was identified as a regulator of alginate biosynthesis and its role in providing c-di-GMP for Alg44 activation will be discussed.[1] Rehm, B.H.A. (2008). In Pseudomonas: Model organism, pathogen, cell factory; ed. Bernd H.A. Rehm; Wiley-VCH, Weinheim, 377-395.[2] Gutsche, J., Remminghorst, U. and Rehm, B.H.A. (2006). Biochimie 88, 245-251.[3] Remminghorst, U. and Rehm, B.H.A. (2006). Appl Environ Microbiol 72, 298-305.[4] Oglesby, L.L., Jain, S. and Ohman, D.E. (2008). Microbiology 154, 1605-15.[5] Merighi, M., Lee, V.T., Hyodo, M., Hayakawa, Y. and Lory, S. (2007). Mol Microbiol 65, 876-95.


Molecular interactions of passenger and transport domains demonstrated with autotransporter chimera of Pseudomonas aeruginosa EstA and Pseudomonas putida EstP Sabrina Schell1, Frank Rosenau1, Karl-Erich Jaeger1 and Susanne Wilhelm1

1Heinrich-Heine-Universität, Düsseldorf, Germany

The esterase EstA from P. aeruginosa is an autotransporter which is required for various cellular functions related to virulence as rhamnolipid production, cell motility and biofilm formation [1]. The homologous esterase EstP from the non-pathogenic strain P. putida KT2440 shows 57 % identity and 67 % similarity to EstA at the level of amino acid sequence. Both proteins are members of the GDSL-hydrolase family and both show esterase activity towards different substrates after expression in Escherichia coli, P. aeruginosa and P. putida. As a unique feature of EstA and EstP, their catalytical active passenger domain remains covalently attached to the translocator

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domain, while for most autotransporters, the enzyme domain is cleaved off after translocation to the bacterial outer membrane. Thus, we assume that the enzyme domain may interact with the β-barrel forming translocator domain. A topological model of the EstA translocator domain supported this assumption, because it showes extended loops exposed to the cell surface which additionally carries a large number of charged amino acids.Here, we report on the construction of chimeras from the autotransporter esterases EstA and EstP by replacing their respective passenger domains. The chimeric proteins were analyzed for secretion and esterase activity in the respective host strains. Indeed, the activity of the chimeras EstANPC and EstPNAC differed for the wild-type proteins EstA and EstP expressed in P. aeruginosa and P. putida. Upon fusion to the heterologous alkaline phosphatase signal peptide from E. coli the secretion efficiency of the chimera EstANPC increased in contrast to poor secretion efficiency of EstPNAC in E. coli. Furthermore, we have overexpressed EstA, EstP and the respective chimeras as inclusion bodies. After purification and refolding, the enzymatic activities were determined using p-nitrophenol esters of different chain length fatty acids as substrates. All constructs showed significantly different enzymatic activities. Our results suggest that specific interactions are required between passenger and translocator domains of an autotransporter to ensure its cellular localisation and correct biological function.

[1] Wilhelm, S. Gdynia, A., Tielen, P., Rosenau, F. Jaeger, K.-E. (2007). The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation. J Bacteriol. 189: 6695-6703


Structural and biochemical characterization of the OprD outer membrane channel familyBert van den Berg1

1UMass Medical School, USA

In pseudomonads and other gram-negative bacteria that do not have general porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. In Pseudomonas aeruginosa, an opportunistic human pathogen, the OprD family has 19 members which are closely related in sequence (45-55% pairwise identity). The structural basis for the substrate specificity of OprD channels is unknown.We have determined X-ray crystal structures of OprD and OpdK, OprD family members implicated in the uptake of basic amino acids and vanillate and related aromatic acids, respectively. The crystal structures reveal that OprD and OpdK have monomeric 18-stranded beta-barrels and are very similar overall. The pore constriction present in the OprD and OpdK structures is novel, and is formed by the inward-folded loops L3 and L7. The pores are lined by a striking, positively charged ladder of basic amino acid residues on one side and an electronegative pocket on the opposite side, features likely to be important for substrate selection. Despite the overall structural similarity of OprD and OpdK, the shape and size of the pores are substantially different for both channels, with the OpdK pore constriction being relatively wide for a substrate-specific channel (~8 Å diameter). Single-channel electrical recordings of OprD and OpdK confirm the difference in pore sizes, and demonstrate that vanillate is a substrate of the OpdK channel. Finally, blue-native gels suggest that both OprD and OpdK form labile trimers in the outer membrane. In summary, the comparison of the OprD and OpdK structures provides the first qualitative insights into the different substrate specificities of these closely related channels. A more extensive characterization of OprD family members may allow development of a "permeability database" against which drugs directed against P. aeruginosa could be screened.


Characterization of the cell wall stress response and recovery through σ22 and its controlled factors in Pseudomonas aeruginosa Lynn F Wood1 and Dennis E Ohman1

1Virginia Commonwealth University Medical Center and McGuire Veterans Affairs Medical Center, Richmond, VA, USA

Chronic pulmonary disease caused by Pseudomonas aeruginosa is associated with strains overproducing alginate due to mutations in mucA. Alginate confers antiphagocytic and microcolony-biofilm properties that are important to pathogenesis. The mucA gene encodes an anti-sigma factor that sequesters alternative sigma factor Sigma22 and prevents expression of its target promoters, which include the algD operon for alginate production. Mutations in mucA cause de-repression of Sigma22 activity, constitutive algD expression and thus alginate production. Previously, we have shown that cell wall stress agents (e.g. D-cycloserine) in MucA+ bacteria also induce algD expression by activating Sigma22 activity. Current in vitro studies showed that periplasmic MucB interacts with MucA as part of the sigma-sequestration complex. We observed that exposure of strain PAO1 to D-cycloserine caused degradation of MucA within just 10 minutes of exposure. This was dependent on AlgW protease (PA4446), which is a DegS homologue. YaeL protease (PA3649) was also required for Sigma22 activation, but not for cleavage of MucA by AlgW. Thus, AlgW and YaeL affect different stages of the regulated intramembrane proteolysis (RIP) of MucA. D-cycloserine showed dose dependent effects on MucA degradation and on Sigma22 dependent transcription. Using a PalgD::lacZ reporter, a kinetic analysis showed that exposure to D-cycloserine (400 µg/ml) resulted in a peak of Sigma22 activity at 2 h and then recovery to basal levels by 5 h. When PAO1 was repeatedly passaged in media containing D-cycloserine for 90 days, no mucoid conversion was seen, suggesting that prolonged exposure to cell wall inhibitors does not select for permanent de-repression of Sigma22. To evaluate changes in the transcriptome due to cell wall stress, we compared short and long-term (i.e., 15 and 60 min) exposures to D-cycloserine using microarray analysis. By subtracting those genes that were activated by D-cycloserine in a mucA mutant and in an algW mutant, we were able to identify the genes of the Sigma22 regulon within the larger D-cycloserine stimulon. A functional analysis of these 293 genes revealed that many were involved in adaptation/protection and outer envelope homeostasis. We are currently performing a mutant analysis of selected genes from the Sigma22 regulon to better understand how P. aeruginosa responds to and recovers from cell wall stress.

P-190 Topic: Cell Surface, Transport and Secretion

The PilM/N/O/P and FimV proteins form an inner membrane complex required for PilQ and secretin stabilityMelissa Ayers1, Hania Wehbi1, Eder Portillo1, Liliana Sampaleanu2, Stephanie Tammam2, P. Lynne Howell2 and Lori L. Burrows1

1McMaster University, Hamilton, 2Hospital for Sick Children, Toronto, Canada

Type IV pili (T4P) are polar appendages that Pseudomonas uses for attachment and twitching motility. Approximately 1% of the Pseudomonas spp. genome is dedicated to the regulation, maintenance and function of T4P. Of these, the pilM/N/O/P and fimV genes

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are required for twitching but their precise functions are unknown. We hypothesize that their products form an inner membrane complex linking the cytoplasmic and inner membrane components to the outer membrane secretin, a multimer of PilQ subunits. PilM is a putative actin-like cytoplasmic protein; PilN, PilO and FimV are putative inner membrane proteins; PilP is a putative inner membrane lipoprotein. Unmarked, nonpolar, mutations in the pilM/N/O/P genes and a transposon insertion in fimV were characterized, and all lacked twitching motility and surface pili. The PilMNOP and FimV proteins stabilize one another, as Western analyses with specific protein antisera show that loss of individual components has a negative effect on the levels of other components of this hypothesized complex. Furthermore, Western analyses with PilQ antisera revealed that the absence of any of these proteins has a negative impact on levels of the T4P secretin. Phenol dissociation of the heat and SDS-resistant secretins showed that this phenotype was likely due to a decreased stability of PilQ monomers, and not to reduced assembly of secretin multimers. To assess whether the complex was stable in the absence of pili, mutants unable to make the major subunit PilA (pilA, rpoN, pilR, pilS) were examined. Unexpectedly, the complex was destabilized in pilA and rpoN mutants, but not in pilR or pilS mutants. Together, these data support our hypothesis that the PilM/N/O/P and FimV proteins form an inner membrane complex that may interact with and stabilize PilQ monomers. This finding contrasts with data from the T4P systems of Neisseria meningitidis and Myxococcus xanthus, where PilQ stability is independent of PilM/N/O/P. Moreover, our data implies that the PilR/S two component system negatively regulates complex stability in the absence of PilA.


The lectin LecB of Pseudomonas aeruginosa interacts with the outer membrane porin OprFKai-Malte Bartels1, Susanne Wilhelm1, Robert E.W. Hancock2, Frank Rosenau1 and Karl-Erich Jaeger1

1Institute for Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Research Centre Juelich, 2Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada

Pseudomonas aeruginosa, an important opportunistic pathogen associated with chronic airway infections, synthesizes two lectins, the galactophilic LecA and the fucose- and mannose-binding LecB, which exert different cytotoxic effects on respiratory epithelial cells [1]. The LecB protein is involved in biofilm formation, pilus biogenesis and proteolytic activity [2, 3]. It is localized in the cytoplasm and binds to carbohydrate ligands in the outer membrane [2]. Now, we have demonstrated that LecB could be released from the bacterial cell surface by treatment of biofilm cells with L-fucose, whereas treatment with D-galactose had no effect, indicating that LecB is cell-surface exposed. Thus, we have analyzed putative interactions of LecB with other P. aeruginosa proteins. LecB and LecB-His6 were expressed and purified using affinity chromatography on mannose agarose. Furthermore, Western blotting with purified LecB revealed the outer membrane porin OprF as a putative LecB ligand. This result was confirmed by binding of OprF to His-tagged LecB immobilized on Ni-NTA agarose, and also by affinity chromatography using LecB bound to mannose agarose. Additionally, the subcellular localisation of LecB was analyzed in an OprF-deficient P. aeruginosa strain. Cell fractionation and subsequent immunoblotting revealed that LecB was no longer detectable in the membrane fraction of the mutant strain, but appeared in the culture supernatant. These findings strongly suggest that LecB is bound to the cell-surface in vivo, where it interacts with the outer membrane porin OprF.

[1] Gilboa-Garber N., Katcoff D. J., and N.C. Garber. (2000) Identification and characterization of Pseudomonas aeruginosa PA-IIL lectin gene and protein compared to PA-IL. FEMS Microbiol. Lett. 29: 53-57.[2] Tielker, D., Hacker, S., Loris, R., Strathmann, M., Wingender, J., Wilhelm, S., Rosenau, F., and Jaeger, K.-E. (2005) The Pseudomonas aeruginosa lectin LecB is located in the outer membrane and involved in biofilm formation. Microbiology 151: 1313-1323[3] Sonawane, A., Jyot, J., and Ramphal, R. (2006) Pseudomonas aeruginosa LecB is involved in pilus biogenesis and protease IV activity but not in adhesion to respiratory mucins. Infec. Immun. 74: 7035-7039.


A novel type six secretion effector secreted by Pseudomonas aeruginosa Abderrhaman Hachani2, Iwona Buchior3, Chantal Soscia4, Joanne Engel3 , Alain Filloux2 and Sophie Bleves1

1CNRS-Mediterranean University, 2Imperial College London, Division of Cell and Molecular Biology, Center for Molecular Microbiology and Infection, South Kensignton Campus, Flowers Building, SW7 2AZ London, United Kingdom, 3University of California San Francisco, Department of Medicine, HSE-407, 513 Parnassus Avenue,San Francisco, CA 94143, USA, 4LISM-IMM-CNRS 31 ch. J. Aiguier, 13402 Marseille cedex 20, France

The pathogenicity of Pseudomonas aeruginosa relies on its ability to secrete numerous proteins in the extracellular medium and to directly inject some toxins in the cytosol of the eukaryotic target cell. The recently identified type six secretion pathway (T6SS) is present in 3 copies in the P. aeruginosa PAO1 genome. In contrast to the HSI-I machinery that was shown to secrete the Hcp-1 protein during chronic infection, no substrate and no biological function has been described so far for the two other systems. We discovered that the second machinery, HSI-II, is responsible for the secretion of a protein we called VgrG2. This secretion is specific since VgrG2 is still released in culture supernatants from mutants of the HSI-I and HSI-III machinery. Moreover, we studied the role of the HSI-II machinery and more particularly of its effector during interaction with host cells. Data on macrophages and epithelial cells will be presented in this poster.


Host cell components influencing the membrane insertion, activation and function of the T3SS translocon in Pseudomonas aeruginosa Yu-Sing Tammy Bohn1, Julien Verove1, Francois Cretin1, Marie-Josèphe Rabiet1, Ina Attree1 and Francois Boulay1

1C.E.A. Grenoble / iRTSV / 17, rue des Martyrs / 38054 Grenoble / France

Pseudomonas aeruginosa is able to directly inject the ExoS toxin into the host cell cytoplasm via the proteineous needle, type III secretion system (T3SS). It has been shown that two proteins, PopB and PopD, which harbour putative transmembrane domains, are essential for the functionality of T3SS. In pursue to identify the so far unknown host factors that can influence the effectiveness of the Pseudomonas T3SS, we generated a FRET-assisted reporter system which allows the monitoring of ExoS translocation. This reporter system was applied to the promyeloid cell line HL60 that is the only cell line described so far to be resistant towards ExoS translocation in the non-differentiated state and

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susceptible upon differentiation. First, we confirmed by FACS analysis and fluorescence microscopy that the T3S dependent ExoS translocation is indeed significantly elevated in differentiated HL60 cells. Second, membrane flotation experiments by sucrose gradient ultracentrifugation suggest that translocons formed by PopB and PopD are inserted into the membrane of HL60 cells irrespective of their differentiation state. Furthermore, we have evidences that the translocon may be targeted and inserted into membrane microdomains called lipid rafts. Consistent with this, recombinant translocon proteins were able to recognize certain lipids including cholesterol and cerebrosides which are enriched in lipid rafts. Taken together, these results suggest that the ExoS translocation into target cells by the P. aeruginosa T3SS may not only rely on the insertion of the PopB/PopD translocon into the host cell membrane, but rather requires further activation or stabilization of the translocon pore by yet unidentified host cell components in the vicinity of its insertion site.


Contributions of residues in the propeptide of elastase from Pseudomonas aeruginosa for their role in foldase activity.Emily Boice1, Efrat Kessleer2 and Dennis E. Ohman1

1Virginia Commonwealth University Medical Center and Veterans Affairs Medical Center, Richmond, Virginia, USA, 2Tel-Aviv University, Sheba Medical Center, Tel-Hashomoer, IsraelPseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase, an M4 metalloendopeptidase. Elastase, encoded by lasB, is first synthesized as a preproenzyme, with a signal peptide, a central 18-kDa propeptide, and 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for proper folding and secretion of elastase, but is ultimately removed and degraded proteolytically. Previous research (McIver, K. S. et al., 2004) identified conserved residues in the propeptide of elastase compared to other M4 protease precursors in nature and showed some among them to be important for the production of active elastase. Here, we further examined the effects of substitutions at conserved residues in the propeptide of plasmid-encoded lasB alleles by expressing them in a lasB mutant. Elastase activity in culture supernatants was quantitated using two highly sensitive fluorescent substrates: Abz-AGLA-p-Nitro-Benzyl-Amide and MOCAc-GSPAFPLA-Lys (Dnp)-D-Arg-NH2. In vitro refolding studies are also being performed to determine the effects of specific substitutions on the foldase activity of the propeptide. Wild-type and mutant propeptides with His6 tags (i.e., mature domain deleted) were overexpressed in E. coli and purified from lysates by affinity chromatography. Elastase (native and affinity tagged) was purified by ion exchange chromatography from culture supernatants of strains constructed to overexpress lasB from plasmids. When wild-type propeptide and elastase as separate proteins were denatured in guanidine-HCl / glycine buffer, mixed and then renatured together to refold elastase, approximately 30% activity was observed, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Our goal is to perform a comprehensive analysis of the conserved residues to determine their role in the foldase activity of the propeptide.


TOL plasmid carriage increases biofilm formation in Pseudomonas putida strainsPaul W D'Alvise1, Barth F. Smets1 and Najoi El Azhari1

1Technical University of Denmark, Department for Environmental Engineering, Lyngby, Denmark

The ubiquitous soil bacterium Pseudomonas putida can metabolize a wide range of natural and synthetic organic compounds, and is a model organism for studying biodegradation, vadoze zone physiology and rhizosphere ecology. Despite its primary niche as a soil and rhizosphere dweller, P. putida is a very poor biofilm former. However, the ability to form biofilms is a prerequisite for a number of industrial and environmental applications, so there is a justified interest in understanding the molecular factors that govern biofilm formation in P. putida. The notion that conjugal plasmids generally enhance their host’s biofilm-forming ability and the increasing availability of systems that exemplify this notion will hopefully allow further investigation of these mechanisms. The TOL plasmid is one of the best studied catabolic plasmids, encoding biodegradation pathways for toluenes and xylenes. The TOL plasmid belongs to incompatibility group P and has a broad host range. Here we present evidence that the TOL plasmid increases biofilm formation in pseudomonad strains and suggest P. putida and the TOL plasmid as a model system for plasmid-enhanced biofilm formation.We assessed biofilm formation by P. putida KT2440 and KT2442, both with and without the TOL plasmid, in a standardized flow chamber setup. Of all strains tested P. putida KT2440 had the weakest initial attachment and after seven days almost no adherent biomass was found. P. putida KT2442 biofilms developed into continuous monolayers with protruding filamentous cell chains. The biofilms of TOL harboring strains were multilayered, and mushroom-shaped microcolonies grew over 100 µm in height after 7 days. Also with the TOL plasmid, P. putida KT2442 formed thicker biofilms than KT2440. Currently, we are addressing whether the often noted increased production of extracellular polysaccharides as a reason for the enhancement of biofilm formation also holds for the TOL stimulation of P. putida biofilm formation. Fluorescence microscopy of cell material stained with Calcofluor White has sofar indicated significantly higher EPS production by the TOL carrying strains.As hypothesized before, we think that promotion of biofilm formation is an evolutionary favored trait of conjugative plasmids. As conjugation rates are higher in biofilms, by this trait conjugative plasmids abet their own propagation.


The Pseudomonas aeruginosa Tad machine involvement in Flp pilus biologySophie de Bentzmann1, Bernard Christophe1 and Bordi Christophe1

1CNRS UPR9027, Marseille, France

P. aeruginosa is the only bacterium known to be able to assemble both type IVa and type IVb pili. Genome mining allowed us to identify a type IVb macromolecular machinery of the Flp subfamily called Tad machine which is widespread in Archaea, Actinobacteria and Gram-negative bacteria, usually as a single copy, but in up to four copies in some species. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are necessary for adhesion to abiotic surfaces, as well as to eukaryotic cells. Type IVb pili are composed of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase before being assembled into a pilus that extrudes through an outer membrane protein, the secretin RcpA. This macromolecular system requires an ATPase TadA, at least for Flp pilus assembly (de Bentzmann et al, 2006). This type IVb system of piliation ressembles the type IVa in some ways, however, the presence within the tad locus of several specific genes encoding proteins with no homologue in the T4P and T2S systems, such as RcpC, RcpB, TadZ and TadG, suggests that Tad machines represent a new type of prokaryotic secretion system. In this study, we first gained an insight into the regulatory mechanism of the tad locus. We showed that the expression of the flp gene occurs late in the stationary growth phase under aerobic conditions. We further demonstrated that the locus is composed of five independent

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transcriptional units. Using transcriptional fusions, we showed that the tad gene expression is positively controlled by the PprB response regulator of the two-component system PprAB. Experiments demonstrated that PprB binds to the five promoter regions of the tad locus and directly controls expression of these genes. We then evaluated the contribution of two genes of this tad locus, tadF and rcpC for Type IVb pili assembly, that encode a putative pseudopilin and a putative protein from a family of bacterial proteins containing two beta-clip domains potentially located in the outer membrane or periplasm, respectively. Deletions of these two genes do not affect Flp production, pilus assembly and Flp-mediated adhesion to abiotic surface in our conditions. However, RcpC post-translationally modifies the Flp pilin subunit in a way that promotes Flp-dependent adhesion to eukaryotic cells through a probable glycosylation. These results suggest that the Flp type IVb pilus is a major system involved in early P. aeruginosa adhesion and biofilm formation. Funded by VLM.


Molecular dissection of Xcp pseudopilins from the Pseudomonas aeruginosa type II secretion systemEric Durand1, Sebastien Alphonse2, Badreddine Douzi2, Mariella Tegoni2, Alain Filloux3, Cedric Bernard2 and Romé Voulhoux1

1Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), UPR9027, IFR88/CNRS, 31 Ch. J. Aiguier, 13402 Marseille Cedex 20, France, 2Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR6098, CNRS, Université de Provence et Université de la Méditerranée, Case 932, 163 Av. de Luminy, 13288 Marseille cedex 9, France , 3Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

Type II secretion system (T2SS), also called Xcp secreton in P. aeruginosa, is a major pathway for the release of virulence factors in the extracellular medium. The Xcp secreton is a macromolecular complex involving 12 proteins (XcpA/GspO, XcpP/GspC to XcpZ/GspM) assembled into the bacterial cell envelope. This secretion apparatus shares common features with the type IV pili (T4P) biogenesis system. Five Xcp proteins (XcpT/GspG; XcpU/GspH; XcpV/GspI; XcpW/GspJ; and XcpX/GspK) possess characteristics very similar to the major type IV pilin. Therefore they have been called pseudopilins. Indeed, the major pseudopilin XcpT/GspG is assembled by the Xcp secreton into a fibrillar structure, the pseudopilus. Understanding the biogenesis process leading to the assembly of such fibrillar structure is paramount to decipher the type II secretion mechanism.Here we report the NMR 3D high resolution structure of XcpT/GspG periplasmic domain (Tp). Tp structure is very similar to the 3D structure of PulG, the GspG-homologue in Klebsiella oxytoca. Both share the classical type IV pilin fold. However, our structure revealed that the once-thought variable region that lies between the N-terminal helix and the β-sheet is in fact highly conserved between the GspG-family members. Moreover, structure-based sequence alignment between GspG-family members revealed that HxcT, belonging to the second T2SS (Hxc) found in P. aeruginosa, presents several key amino-acid changes which might explain why those two major P. aeruginosa pseudopilins are not exchangeable. Here, we present the results obtained after hxcT site directed mutagenesis and discuss the structure-function significance of the key residues identified.In order to understand the precise involvement of minor pseudopilins (XcpU-XcpX) in type II secretion and pseudopilus biogenesis, we cloned, expressed and purified their periplasmic domains. Then, we used different techniques such as BIAcore, NMR chemical shift, chemical cross-linking, and affinity co-purification to study the protein-protein interactions network among the Xcp pseudopilins. Here we present the results of this systematic and complete study. Notably, we revealed the presence of an un-previously described quaternary complex between minor Xcp pseudopilins. Our work, together with previously described pseudopilin sub-complexes, paves the way to understand the molecular scaffold behind the pseudopilus biogenesis process.


ExsD inhibits the DNA-binding activity of ExsA, the key regulator of the Pseudomonas aeruginosa type III secretion systemSylvie ELSEN1, Julie Thibault2, Eric Faudry2, Christine Ebel3 and Ina Attree2

1CNRS - LBBSI, 2LBBSI, iRTSV, CEA-Grenoble, 17 rue des Martyrs 38054 Grenoble cedex 9, France, 3LBM, IBS, CEA-Grenoble, 41 rue Jules Horowitz 38027 Grenoble, France

Like several gram-negative bacteria, the opportunistic pathogen Pseudomonas aeruginosa harbors a type three secretion system (T3SS) that allows the injection of several toxins directly into the eukaryotic host cells. The bacterium utilizes complex signalling pathways to modulate T3SS, which are not entirely deciphered. However, it is well established that all T3SS genes are under the direct control of ExsA that is absolutely required for their transcription.The ExsA protein belongs to the AraC/XylS family of transcriptional regulators, which are characterized by significant homology within a DNA-binding domain containing two motifs Helix-Turn-Helix. These proteins are known to regulate diverse bacterial functions including catabolism, response to stress and virulence. Most of them possess an unrelated regulatory N-terminal domain that may be involved in effector binding and dimerization processes. Whereas effectors are usually small molecules like sugars that activate the transcription factor, few AraC/XylS-like proteins were found to be regulated by protein ligands, either positively or negatively. The interactions between an AraC-like protein and its inhibitory ligand were not characterized yet in term of complex formation (domain involved, oligomeric state) as well as of inhibitory mechanism (inhibition of DNA-binding and/or activation of RNA polymerase).We have investigated the mechanism of ExsA inhibition by its known ligand, the anti-activator ExsD. We demonstrated that ExsD does not bind to the ExsA-target sites on DNA but its presence is sufficient to inhibit ExsA transcriptional activation in a heterologous system. After coproduction of the two proteins, we purified the ExsA/ExsD complex, and analytical ultracentrifugation evidenced a 1:1 stochiometry. When in interaction with ExsD, ExsA is not able to bind to its specific target promoters anymore. Therefore we propose a model in which, under non inducing conditions, the anti-activator ExsD sequesters ExsA and blocks its binding to DNA sites, preventing type three secretion gene transcription.


Impact of antibiotic stress on Pseudomonas aeruginosa lipopolysaccharideJulia Garbe1, Herbert P. Schweizer2, Joseph S. Lam3, Michael Hogardt4 and Max Schobert1

1Technische Universität Braunschweig, Institute of Microbiology, Spielmannstraße 7, 38106 Braunschweig, Germany, 2Colorado State University, Department of Microbiology, Immunology and Pathology, Fort Collins, CO 80523-1682, USA, 3University of Guelph, Department of Molecular and Cellular Biology, Guelph, Ontario, Canada N1G 2W1, 4Max von Pettenkofer-Institut, Außenstelle Großhadern, Marchioninistrasse 17, 81377 München, Germany

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The opportunistic human pathogen Pseudomonas aeruginosa typically causes diseases in patients with a compromised immune system. P. aeruginosa is characterized by its intrinsic antibiotic tolerance but also by its ability to develop high-level resistance during antibiotic treatment. Several well-characterized factors as efflux pumps or an impermeable outer membrane are known to contribute to an intrinsic antibiotic tolerance. Moreover, a number of mutations, which further increase antibiotic tolerance, have been identified.Clinical non-CF isolates of P. aeruginosa characterized by a high antibiotic tolerance attracted our attention since these strains contain a modified, highly truncated lipopolysacchride (LPS). We investigate if this truncated LPS contributes to an increased antibiotic tolerance. First, we characterized these clinical isolates and established an in vitro phage based system to screen for the emergence of these LPS variants during antibiotic treatment. The clinical isolates with the truncated LPS contained a mutated MexR repressor. This indicates that the increased antibiotic tolerance is a result of an overexpressed efflux pump. Moreover, when we determined antibiotic tolerance of several P. aeruginosa PAO1 mutants containing different versions of a truncated LPS no increased antibiotic tolerance was detected. These results indicated that the truncated LPS does not directly contribute to an increased antibiotic tolerance e.g. by decreasing the diffusion or impairing uptake of the antibiotic.However, our in vitro phage based system clearly showed that the same type of mutants with a truncated LPS occurs even during antibiotic treatment in vitro - in combination with a mutation of the MexR repressor. But again the truncated LPS does not contribute directly to an increased antibiotic tolerance. However, we noticed that the same mutants were more tolerant to oxidative stress. It was shown that antibiotics cause oxidative stress in bacteria by stimulation of the Fenton reaction. We could demonstrate that the modified LPS increases tolerance to the secondary effects of antibiotics, oxidative stress. Currently we are investigating the role of modified LPS in the tolerance to oxidative stress and identify the mutation by complementation with a cosmid library screen. First results show that this mutation is located in the large core oligosaccharide cluster that includes the genes PA5001-PA5012 of the P. aeruginosa PAO1 genome.


Interaction between the type III secretion system of Pseudomonas aeruginosa and Yersinia pestis and the human immune system.Claire Sophie Gendrin1, Hugues Lortat-Jacob2, Andres Palencia3, Ina Attrée-Delic4 and Andrea Dessen5

1Laboratoire des protéines Membranaires, 2LEM, Institut de Biologie Structurale, 3EMBL Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France, 4iRTSV CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 9, 5LPM, Institut de Biologie Structurale, France

Pseudomonas aeruginosa utilizes a type III secretion system (TTSS) to inject toxins into host cells to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found at the tip of the needle. PcrV and its Yersinia homologue LcrV show 42% sequence identity, the highest conservation being at the C termini (Nanao et al. 2003). Both PcrV and LcrV have been shown to be protective antigens against infection with their respective pathogens (Sawa et al., 1999; Franck et al., 2002), and LcrV is currently in phase II trials as the principal agent of a protective vaccine against plague.If LcrV is an immune target for the human body to block infection, it is also able to manipulate the immune sytem, i.e. by stimulating the expression of interleukin-10. Recently, it has been shown that, with cellular biology techniques, LcrV could also interact with interferon-γ (IFN-γ) bound to its receptor (Abramov et al., 2007).In this study, we characterized the interaction of the pathogenic proteins LcrV and PcrV with human IFN-γ. Using the Biacore system, we studied the kinetics and affinity of these interactions. The use of specific IFN-γ mutants allowed identification of the residues of IFN-γ involved in the interaction. Potential inhibitors (Sarrazin et al., 2005) of the interaction were tested: three of them bound IFN-γ with a high affinity, and thus prevented the formation of the complex LcrV-IFN-γ. Microcalorimetry was used as an alternative method to measure the affinity between LcrV and IFN-γ, and yielded a Kd of 277 nM. Once completed, these promising results could yield to the development of a new class of antibiotics.


The ferrichrome iron uptake pathway in Pseudomonas aeruginosa and Escherichia coli: their similarities and differences. Mélissa Hannauer1, Yaniv Barda2, Abraham Shanzer2 and Isabelle Schalk3

1Métaux et Microorganismes : Chimie, Biologie et Applications. FRE3211, CNRS-Université de Strasbourg, ESBS, 2Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, 3Métaux et Microorganismes : Chimie, Biologie et Applications. FRE3211, CNRS-Université de Strasbourg, ESBS, Blvd Sébastien Brandt, F-67413 Illkirch, Strasbourg, France.

To acquire iron, Pseudomonas aeruginosa secretes two major fluorescent siderophores, pyoverdine and pyochelin. This bacterium is also able to use other iron chelators present in the environment like ferrichrome, a siderophore produced by the fungi Ustilago spherogena. In the present work, we investigated the ferrichrome iron uptake pathway using a fluorescent labelled ferrichrome. This molecule is fluorescent in the absence of iron, and once loaded with ferric iron, the fluorescence is completely quenched. This property was used to follow the iron uptake by the ferrichrome in P. aeruginosa cells, the dissociation of the ferrichrome iron complex inside the cells and afterwards the siderophore recycling into the extracellular medium. The cells used for this study were unable to produce pyoverdine and pyochelin. We have shown that the outer membrane transporter involved in this iron uptake is FiuA. When this protein was mutated, no more ferrichrome-Fe uptake was observed. Once the iron was released in the bacteria, fluorescent ferrichrome was recycled into the extracellular medium. We have clearly shown that this recycled ferrichrome was still able to chelate ferric iron with the same kinetic as ferrichrome (ferrichrome, which has never been used by the bacteria) and was also able to transport again efficiently 55Fe into the cells. This result lead us to conclude that ferrichrome recycled by P. aeruginosa seems not to be modified like in E. coli and that the ferrichrome iron uptake pathway in P. aeruginosa and in E. coli present clearly differences in step of iron release from the siderophore. However, further studies are needed to confirm this hypothesis.

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AlgE and AlgK are the outer membrane components of a novel exopolysaccharide secretion complexP. Lynne Howell1, Carrie-Lynn Keiski1, Mirela Neculai 2, John Whitney1, Joel Weadge2, Patrick Yip2, Danny Li2, Christine Bear1, Mike Harwich3, Lynn Wood3, Dennis Ohman3, Howard Robinson4 and Lori Burrows5

1The Hospital for Sick Children/University of Toronto, 2The Hospital for Sick Children, 3Virginia Commonwealth University, 4Brookhaven National Laboratory, 5McMaster University, Canada

Introduction: The exopolysaccharide alginate is the major component of Pseudomonas aeruginosa biofilms infecting the lungs of cystic fibrosis patients. To date, ten proteins have been implicated in alginate polymerization, modification and export. These proteins are believed to form a multi-protein complex that spans the cell envelope and facilitates export of the polymer. Our studies on the alginate secretion complex have focused on the putative porin, AlgE, and the outer membrane lipoprotein, AlgK. Results: Complementation and subcellular fractionation studies have shown that AlgE is required for alginate secretion, and that AlgK plays an important role in localizing AlgE to the outer membrane; a finding that suggests that the two proteins may interact directly. The structure of AlgK has been determined to 2.5 angstrom resolution and reveals an all alpha-helical solenoid protein with 9.5 tetratricopeptide-repeat like (TPR) protein-protein interaction motifs arranged in a right-handed superhelix. Electrophysiological and gel-shift SDS-PAGE assays using over-expressed, refolded AlgE have shown that this protein is an anion selective porin, consistent with its role in alginate export. A direct interaction between AlgK and AlgE has been observed using an in vitro protein pull-down experiment. Conclusions: Our data establish that AlgK and AlgE are critical for alginate secretion, and that the two proteins interact directly, likely via N-terminal TPR motifs of AlgK. These proteins appear to be the outer membrane components of a novel type of secretion complex that differs from currently defined canonical capsular polysaccharide secretion systems. Furthermore, our bioinformatics analyses provide strong evidence that an outer membrane AlgE-AlgK-like secretion complex is involved in the production of other bacterial exopolysaccharides, including cellulose and poly-β-1,6-N-Acetyl-D-glucosamine.


Structural studies of components of the type III secretion pore in Pseudomonas aeruginosaViviana Job1, Pierre-Jean Mattei1, David Lemaire2, Eric Faudry3, Ina Attree3 and Andrea Dessen1

1Institut de Biologie Structurale Grenoble, 2IBEB/SBVME CEA Cadarache, 3iRTSV CEA Grenoble, France

Pseudomonas aeruginosa, as many other pathogenic bacteria, possesses on its surface a secretion system that allows it to inject toxins directly into the cytoplasm of eukaryotic cells. This system, called Type III Secretion System (TTSS), is a multimeric complex composed mostly of membrane proteins, and can be divided into 3 parts: a basal structure that anchors the system to the bacterial membranes, a needle that contacts the host cell, and a translocation pore that crosses the host membrane. Secretion of the pore-forming proteins (PopB and PopD) is the first event which is induced by contact between bacterial and host cells. Pore formation is an absolute requirement for toxin injection into the host cytoplasm. When the system is not induced, PopB and PopD remain in the cytoplasm of Pseudomonas complexed to their common chaperone, PcrH, whose role is to stabilize the two membrane proteins. Our group is interested in understanding the interactions between the two Pop membrane proteins and PcrH. We have shown that each Pop molecule exists as a 1:1 complex with PcrH, and have solved the high resolution crystal structure of a truncated form of PcrH in apo form. PcrH displays a typical TPR (Tetratricopeptide) domain fold which is similar to the one observed for SycD, its homologue from Yersinia. The close conservation of the folds between PcrH and SycD reflects the functional conservation of this chaperone in different bacteria. Efforts are currently underway in our group towards obtaining the structure of PopD:PrcH complexes; the regions of interaction between PcrH and PopD were also studied using limited proteolysis and structural studies of the truncated forms are under investigation. The translocators are key elements for the functionality of the TTSS and toxin injection. Structural information of these proteins alone or associated to their partner could be used for the development of antibacterial molecules that could prevent the formation of the translocation pore, subsequently blocking infection by a variety of bacterial pathogens.


The type II secretion-system of Burkholderia glumae influences biotenside production and biofilm formationJanina Knorr1, Silke Isenhardt1, Susanne Wilhelm1, Karl-Erich Jaeger1 and Frank Rosenau1

1Heinrich-Heine-Universität, Institut für Molekulare Enzymtechnologie, Forschungszentrum Jülich, Germany

Burkholderia glumae, formerly known as Pseudomonas glumae, is a phytopathogenic beta-proteobacterium with a significant biotechnological potential. B glumae possesses a Gsp (general secretory pathway) protein secretion system which is homologous to the type II or Xcp-system of the human pathogen Pseudomonas aeruginosa.We have constructed two Gsp-mutants by inserting an antiobiotic resistance cassette into the gene gspD and the operon gspDEF and we have studied different phenotypes of the corresponding B. glumae mutants.The B. glumae wild-type genome contains genes homologous to the rhamnolipid genes rhlA, rhlB and rhlC of P. aeruginosa. Under specific growth conditions we observed that biosurfactant was produced and transcript was detectable for all genes. Interestingly, both gsp-mutants showed a significantly reduced production of biosurfactant and the amount of transcript was sharply reduced or even absent.B. glumae wild-type was able to form biofilms in minimal medium, whereas both mutants did not when grown under the same conditions. However, biofilm formation of the mutants could be rescued by addition of spent cell-free growth medium obtained from the wild-type strain. Initial analysis of medium components revealed that the "biofilm-inducing factor" had a molecular mass of > 50 kDa. Subsequent size exclusion chromatography allowed to determine an apparent molecular mass of 290 kDa and suggested that the "biofilm inducing factor" is a protein complex. The proteins involved are currently identified by mass spectrometry.

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Characterization of Pseudomonas aeruginosa PilF and its role in type IV pili secretin assemblyJason Koo1, Stephanie Tammam1, Shao-Yang Ku1, Tim Tang1, Liliana Sampaleanu2, Lori L. Burrows3 and P. Lynne Howell2

1University of Toronto, 2Hospital for Sick Children, 3McMaster University, Canada

Introduction: Pseudomonas aeruginosa is a deadly opportunistic Gram-negative pathogen of immunocompromised, cancer, HIV, transplant and burn patients. A key virulence factor used by P. aeruginosa to establish infection is its Type IV pili (T4P). As P. aeruginosa without T4P are avirulent, these organelles make attractive targets for the development of novel therapeutics. Amongst the proteins essential for T4P assembly is the predicted periplasmic lipoprotein, PilF. Although the specific mechanism of PilF action is unknown, studies of PilF homologues in other systems suggest that it interacts with the outer membrane secretin PilQ, through which the pili exit the cell. Methods & Results: Cellular fractionation studies show that PilF is found predominantly in the outer membrane and that its absence prevents the outer membrane localization and multimerization of PilQ. T4P-mediated twitching motility and surface piliation is abolished in the pilF mutant. Mutation of the PilF canonical lipidation site (Cys18) releases the protein into the periplasm as a soluble protein and results in the formation of PilQ multimers in both the inner and outer membranes. Our 2.0 Å X-ray structure of PilF reveals a predominantly helical protein with 6 tetratricopeptide-like (TPR) protein-protein interaction motifs arranged in a bi-layered crescent shape. Site-directed mutagenesis of a conserved N-terminal cluster identified by bioinformatics and structural analysis shows a hydrophobic groove is important for PilF function. Conclusions: Our in vivo functional characterization and crystal structure support the hypothesis that PilF interacts with PilQ in the T4P assembly complex. The TPR motifs of PilF likely mediate the interaction(s) that cause the outer membrane localization and multimerization of PilQ. Experiments to demonstrate a direct PilF-PilQ interaction and to identify the residues involved are in progress.


Role of the c-terminal α-helix of PelC for function and localisation of the proteinKarolina Magdalena Kowalska1, Perrine Vasseur2, Rome Voulhoux2, Chantal Soscia2 and Alain Filloux1

1Imperial College, Centre for Molecular Microbiology and Infection, South Kensington Campus, London SW5 9SX, 2CNRS, 31 chemin Joseph Aiguier 13402 Marseille Cedex, France

Pseudomonas aeruginosa is a human opportunistic pathogen, able to cause severe acute and chronic infections. Among the factors important for chronic bacterial colonisation are exopolysaccharides (EPS). EPSs are involved in the establishment of a particular bacterial organisation called biofilm. It has been shown previously that the pel gene cluster is required for biofilm formation and encodes seven components (PelA-G) that take part in the production of a glucose-rich exopolysaccharides (Friedman & Kolter, Vasseur).In the present study we have concentrated our work on PelC, a key protein of the Pel complex. We first showed that PelC is an outer membrane lipoprotein (Vasseur). Furthermore, we proposed that PelC is a member of an unconventional family of outer membrane proteins, of which Wza is an archetype. Wza structure showed that it possesses an amphiphatic alpha-helix at its C-terminus (Drummesmith, Whitfield). Oligomerization of Wza through the amphipatic helix allows the insertion in the outer membrane and the formation of an alpha-barrel through which exopolysaccharide could be exported to the bacterial cell surface. This characteristic is highly original and radically different as compared to the well-known beta-barrel found in outer membrane porins for example.We predicted that PelC, such as Wza, is having an amphipatic alpha helix at its C terminus. In order to test the amphiphatic nature of this putative helix, its importance in protein functionality and in outer membrane insertion, we engineered a series of PelC derivatives. We constructed PelC variants deleted for the alpha helix or including amino acid substitutions that disrupt its amphipatic nature. We tested the functionality of the PelC variant in biofilm assay experiments. We analysed their subcellular localisation, and more particularly the outer membrane localisation using sucrose gradient fractionation. Our experiments showed that introducing a residue with a negative charge within the hydrophobic side of the helix led to non-functionality, whereas conservative substitution affected neither the function nor the localization of PelC. Furthermore, we showed that deleting the alpha helix disrupted the functionality of the protein even though PelC could remain anchored in the outer membrane via its lipid anchor. However, in this case PelC is not anymore an integral outer membrane protein, and cannot be accessed from the bacterial cell surface as seen by immuno-fluorescence microscopy.


Regulation of two glycosyltransferase genes, migA and wapR, involved in the synthesis of the capped and uncapped core in lipopolysaccharide of Pseudomonas aeruginosaJoseph S. Lam1, Sarah Ostler1 and Dana Kocíncová1

1University of Guelph, Dept. of Molecular and Cellular Biology, 50 Stone Road E., Guelph, ON, N1G 2W1, CANADA

Lipopolysaccharide (LPS) is a virulence factor of Pseudomonas aeruginosa that is composed of lipid A, core oligosaccharide (OS) and a long-polysaccharide (PS) chain (O-antigen). Most cystic fibrosis clinical isolates of P. aeruginosa produces LPS of the rough phenotype, completely devoid of the O-antigen. Our group has identified two genes, wapR (PA5000) and migA (PA0705), which encode putative rhamnosyltransferases responsible for the synthesis of two distinct core OS, termed capped and uncapped core OS, respectively. The transfer of a rhamnose residue in an alpha-1-3-linkage to the backbone of the core by WapR provided a receptor for O-antigen attachment, while the transfer of rhamnose to the core backbone in an alpha-1-6-linkage produces an uncapped core. Understanding the regulation of these two genes, migA and wapR, is important to reveal the role of each of these genes in the proportion of O-antigen capping in P. aeruginosa LPS. Our group recently revealed that complementation of knockout mutants of migA and wapR with the corresponding wild type (WT) gene on a replicative plasmid was insufficient to return the LPS phenotype to WT. Specifically, these complemented strains showed a shift in the ratio of capped versus uncapped LPS that was dissimilar to that of WT. We hypothesize that these observed phenotypes are attributed to gene expression and dosage issues with the multi-copy complementation vector being used. To test this, the knockout strains of these two genes were complemented with a single copy of migA or wapR under the control of its natural promoter in a mini-CTX integrative vector system and LPS production from each of the complemented mutant was restored to a WT-like phenotype, indicating that migA and wapR gene expression levels can influence LPS phenotype and potentially O-antigen

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capping in P. aeruginosa. MigA is regulated by the RhlI/RhlR quorum-sensing (QS) system, while no information concerning the regulation of wapR is yet available. Potential las-like boxes were found upstream of the wapR gene, indicating that wapR may be regulated by QS. The LPS from the QS-deficient strains were analyzed and revealed only slight differences in the ratio between capped and uncapped core LPS between WT and the QS-deficient strains. Quantitative real-time PCR experiments are currently underway in order to ascertain whether wapR is indeed regulated by QS.


Prevalence of efflux pumps overexpression among ciprofloxacin low-level resistant clinical strains of Pseudomonas aeruginosa may not improve high-level resistant mutants’ emergence.Catherine Llanes1, Lucie Vettoretti1, Isabelle Patry2, Marion Dirand1 and Patrick Plésiat1

1Faculty of Medicine, 2CNR Résistance aux Antibiotiques : Pseudomonas aeruginosa, Besançon, France

Low-level resistance to highly potent fluoroquinolones (FQs), such as ciprofloxacin and levofloxacin, may impair the efficacy of single therapy with these molecules in Pseudomonas aeruginosa infections (Dupont et al., JAC 2005). In this study, we systematically investigated the resistance mechanisms to FQs of 86 P. aeruginosa clinical strains exhibiting low-level resistance to ciprofloxacin (MICs of 0.25 to 2 mg/L). As expected, a high prevalence of efflux overproducing mutants (86%) was recorded in these strains. In addition to the widely distributed MexAB-OprM and MexXY/OprM overproducers, this study showed a quite high prevalence of MexEF-OprN mutants (11 isolates). In contrast, MexCD-OprJ mutants were less frequent (2 isolates). None of the other efflux systems (MexGHI-OpmD, MexPQ-OpmE, PmpM and PA1541) known to accomodate FQs was found to be overexpressed in the clinical strains. Surprisingly, 7/86 isolates harborded target alterations in GyrA or GyrB with 1/7 concomitant to MexAB-OprM derepression. In 6/86 strains, none of the investigated mechanisms was detected. The accumulation of several (up to three) mechanisms in individual isolates raises the question of the interplays between these mechanisms and their contribution to emergence of higher resistance levels to FQs. To assess whether upregulation of efflux may promote the development of second-step mutants showing strong resistance to ciprofloxacin, we compared the mutant rates on ciprofloxacin 2 and 4 mg/L from 10 wild type strains of P. aeruginosa and 10 efflux overproducers. In contrast to what had been anticipated, our results showed that efflux upregulation is not a prerequisite to the development of FQs highly resistant strains.


Structural studies of the T3SS translocation pore of P. aeruginosaPierre-Jean Mattei1, Andreas Forster2, Viviana Job1, Daphna Fenel1, Guy Schoehn1, Ina Attree3 and Andrea Dessen1

1Institut de Biologie Structurale Grenoble, 2Imperial College London, 3iRTSV CEA Grenoble, France

During acute infection, Pseudomonas aeruginosa employs a remarkable weapon, the type III secretion system (T3SS), for secretion and translocation of toxins directly into the eukaryotic host cell. These toxins have dramatic effects on infected cells, leading to cytoskeleton rearrangement and oncosis. The T3SS is composed of three main structural parts: a basal body anchored to the bacterial double membrane, a needle formed by a unique, polymerized protein, and a pore which is inserted into the target membrane. The latter structure is composed of two hydrophobic proteins (PopB and PopD) that must be secreted through the interior of the polymerized needle and form oligomers upon target membrane binding. Whilst in the bacterial cytoplasm, however, PopB and PopD remain associated to their common chaperone, PcrH, which prevents their aggregation and/or oligomerization, which could be lethal for the cell. Our group is particularly interested in PopB, a 400-residue membrane protein carrying two potential transmembrane regions which has been shown as essential for T3SS-related bacterial cytotoxicity. We have succeeded in purifying PopB in soluble form, in complex with its chaperone in large amounts (more than 100 mg). The complex can be separated at acidic pH in the presence of different detergents and lipids. Negative staining electron microscopy experiments have revealed that PcrH-free PopB purified in the presence of specific detergents forms ring-like structures with an approximate exterior and interior diameters of 80 and 30 Å, respectively, which is comparable to previous observations in the presence of liposomes. We provide evidence that these structures could represent PopB hexamers, as has been seen for the homologous protein SipB in Salmonella spp. This work represents the first biophysical characterization of a T3SS translocation pore and provides important insight into the structure of this important molecule.


Protein-protein and protein-DNA interactions of the Cgr proteins regulating phase-variable expression of the cupA gene clusterHeather Rose McManus1 and Simon Dove1

1Children's Hospital Boston, USA

The cupA gene cluster of Pseudomonas aeruginosa encodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. In P. aeruginosa the control of cupA gene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. This regulation is in part mediated by MvaT-mediated repression of three genes (cgrABC), all of which are required for cupA expression. The mechanism by which the cgr genes positively regulate cupA expression is poorly understood. Here we show that CgrC and CgrB can form homomers, and that CgrC and CgrA interact with one another directly. Furthermore, we show that in vivo the ParB-like protein CgrC associates with the cupA promoter region. Our findings suggest that a complex formed by CgrA and CgrC regulates expression of the cupA gene cluster by binding directly to the cupA promoter region. Our results provide a framework for understanding how phase-variable expression of the cupA gene cluster might be achieved.


Elucidating the pathway of RsmA-mediated regulation of the type three secretion system in Pseudomonas aeruginosa.Hazel F O'Connor1, Marlies Mooij1, Heidi Mulcahy2, Claire Adams1 and Fergal O'Gara1

1BIOMERIT Research Centre, Dept. of Microbiology, University College Cork, Cork, Ireland, 2Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, T2N 4N1, Canada

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The Type Three Secretion System (TTSS) is one of the main virulence mechanisms contributing to the pathogenesis of Pseudomonas aeruginosa during acute infection. The system facilitates the delivery of bacterial effector toxins, including ExoS, ExoU, ExoT and ExoY, directly into host cells, leading to the modulation of several immune-related regulatory pathways in the host (O’Grady et al., 2007). The expression of both the secretory apparatus and effector molecules of the TTSS is coordinated by a complex regulatory cascade composed of an array of sensors and regulators. Recently, we have shown that the post-transcriptional regulator RsmA positively regulates the TTSS and in turn, cellular cytotoxicity in in vitro studies (Mulcahy et al., 2006). RsmA also plays an important role in murine infection in vivo, whereby RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia but inactivation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice (Mulcahy et al., 2008). This study aimed to investigate the mechanism by which RsmA regulates the TTSS. Results suggest that this occurs indirectly via transcriptional regulators. MvfR is a LysR-type transcriptional regulator, which controls the transcriptional regulation of multiple intercellular signalling molecules, including the Pseudomonas quinolone signaling (PQS) system. Previous published data suggests that MvfR post-transcriptionally regulates rsmA (Deziel et al., 2005), however our work demonstrates that RsmA post transcriptionally regulates mvfR. Here, we present the regulatory interaction between RsmA and MvfR and the role of MvfR in RsmA-mediated regulation of the TTSS.References:O'Grady E, Mulcahy H, Adams C, Morrissey JP, O'Gara F. Nat Rev Microbiol. 2007 May; 5(5):337-41.Mulcahy H, O'Callaghan J, O'Grady EP, Adams C, O'Gara F. Infect Immun. 2006 May; 74(5):3012-5.Mulcahy H, O'Callaghan J, O'Grady EP, Maciá MD, Borrell N, Gómez C, Casey PG, Hill C, Adams C, Gahan CG, Oliver A, O'Gara F. Infect Immun. 2008 Feb;76(2):632-8Déziel E, Gopalan S, Tampakaki AP, Lépine F, Padfield KE, Saucier M, Xiao G, Rahme LG. Mol Microbiol. 2005 Feb; 55(4):998-1014.


Regulatory elements of a σN-dependent Pseudomonas sp. strain ADP operon encoding a putative ABC-type transporterAna I. Platero1, Manuel García-Jaramillo1, Eduardo Santero1 and Fernando Govantes1

1Centro Andaluz de Biología del Desarrollo/Universidad Pablo de Olavide, Sevilla, Spain

The orf98-97-96-95-94 genes from the Pseudomonas sp. ADP atrazine degradative plasmid pADP-1 encode the components of a putative ABC-type transporter. Here we describe the trans- and cis-acting elements involved in expression and regulation of all five genes. Our results show that orf98, orf97, orf96, orf95 and orf94 form an operon that is transcribed from a single promoter located upstream from orf98. The Porf98 promoter is sigma-N-dependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by the product of the gene located immediately upstream from orf98, atzR. AtzR is a lysR-type transcriptional regulator (LTTR) involved in activation of the cyanuric acid degradative operon atzDEF. A combination of mutational analysis in vivo and primer extension has allowed us to locate the Porf98 promoter and map the transcriptional start site. Similarly, we have used deletions and site-directed mutagenesis together with in vivo expression assays to locate the NtrC and AtzR binding sites, and address their functionality. Our in vivo expression results, along with the arrangement of the promoter elements suggest a regulatory model in which NtrC activates Porf98 transcription via DNA looping and AtzR antagonizes NtrC function by interfering with the activation process.


Co-chaperon interactions in stabilization of the type III needle component PscF of Pseudomonas aeruginosaSophie Plé1, Yung-Sing Wong2, Andréa Dessen3 and Ina Attrée1

1CEA Grenoble/iRTSV/LBBSI - 17, avenue des Martyrs - 38054 Grenoble, 2UJF/DPM/LCBO - Bâtiment E - 38041 Grenoble, 3IBS - 41, rue Jules Horowitz - 38027 Grenoble, France

The Type III secretion (T3S) system plays a key role in the pathogenicity of Pseudomonas aeruginosa and is employed to inject toxins directly into the cytoplasm of target cells. It is composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which the toxins travel. The T3S needle is formed by polymerization of its main component PscF which needs two cytoplasmic partners, PscE and PscG, to stay in a monomeric state and to maintain its secretion prone conformation. Recently, the crystal structure of the PscE-PscF-PscG complex regulating the T3S needle formation has been solved, thus we pursued to examine the implication of the protein-protein interfaces in Pseudomonas pathogenicity by using knock-out mutants deficient in PscE and PscG. First results revealed that both mutants are non-cytotoxic towards macrophages, lack PscF, and are unable to export in trans encoded PscF. Failing to co-purify PscG with PscF, we suggest that the role of PscE may be to consolidate the PscG:PscF dimer thus giving room for speculation on PscG to be the more important PscF-chaperon within the trimeric complex. As a matter of fact, when overproduced, the PscG:PscF complex is able to compensate the lack of PscE. In accordance to this, membrane purification by sucrose-gradient ultracentrifugation using strains overproducing PscF and PscG but lacking of PscE showed evidences that PscG may play a role in the export of PscF through the membrane.Another orientation of our work on PscG was to focus on its interaction surface with PscE that is of 1321Å². Using site-directed mutagenesis, we defined on both proteins the first interaction hot-spots involving three to six hydrophobic amino acids, Then in pursue to inhibit the complex formation, molecules from the Prestwick Chemical Library were screened by ELISA test. Currently, we work on the chemical modification of the selected hits to strengthen their affinity with the protein targets and to find new drugs for an alternative antibiotic therapy.

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An original mechanism for the secretion of a patatin-like protein by Pseudomonas aeruginosaRichard Salacha1, Filip Kovacic2, Jan Tommassen3, Alain Filloux4, Romé Voulhoux5 and Sophie Bleves5

1CNRS, 2Institute for Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf at FZ-Juelich Stetternicher Forst, D-52426 Juelich, Germany, 3Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands, 4Centre for Molecular Microbiology and Infection, Division of Cell and Molecular Biology, Faculty of Natural Science, Flowers Building, Room 1.42, South Kensington Campus, Imperial College London London SW7 2AZ, UK, 5Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM) UPR9027-CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France

The pathogenicity of Pseudomonas aeruginosa is mainly due to its ability to secrete a great number of proteins in the extracellular medium or to directly inject some toxins in the cytosol of the target eukaryotic cell. Remarkably, these proteins have eukaryotic domains, usually in order to interfere with host cellular processes. For instance the active domain of the ExoU cytotoxin [1] of P. aeruginosa PA103 strain is homologous to patatins. Patatins are storage glycoproteins from potato tubers that possess a phospholipase A2 activity and that are involved in defense against stress and pathogen infections. Genes encoding for Patatin-Like-Proteins (PLP) have been identified in several genomes of pathogenic or symbiotic bacteria [2], which suggests how important such domains in bacteria-host interaction are. Looking for PLPs in the proteome of P. aeruginosa strain PAO1, we found one interesting candidate, named PlpD. In silico caracterisation of PlpD revealed that PlpD shares characteristics of both AT (AutoTransporter) and TPS (Two-Partner Secretion), the two branches of the Type V Secretion System [3] (T5SS). AT comprise a family of secreted proteins able to translocate themselves across the outer membrane. The TPS pathway involves 2 protagonists: the secreted protein (TpsA) capable to cross the outer membrane thanks to a protein (TpsB). In PlpD, as in AT, a classical signal peptide precedes a passenger domain with an enzymatic activity (the catalytic dyad of β-domain is typical from TpsB the PLP family), but the C-terminal PlpD protein (including remarkably a POTRA -polypeptide-transport-associated- motif). We showed that the PLP domain of PlpD was secreted in the extracellular media after cleavage from the β-domain that stayed in the outer membrane. PlpD is clearly the first member of a new family of T5SS secreted protein that we proposed to call POTRAnsporter.[1] Sato and Franck (2004) Mol Microbiol 53:1279[2] Banerji and Flieger (2004) Microbiology 150:522[3] Henderson et al (2004) Microbiol Mol Biol Rev 68:692


Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complexLiliana Sampaleanu1, Cathy Horvatin1, Jason Koo1, Stephany Tammam1, Melissa Ayers2, Jeffrey B Bonanno3, Steve C Almo4, Stephen K Burley5, Lori L Burrows2 and P Lynne Howell1

1Hospital for Sick Children, Canada, 2McMaster University, Canada, 3New York Research Centre for Structural Genomics, USA, 4Albert Einstein College of Medicine, USA, 5Eli Lilly Co., USA

Introduction: Pseudomonas aeruginosa Type IV pili (T4P) are virulence factors involved in chronic infections in cystic fibrosis and immunosuppressed patients. Absence of T4P prevents colonization suggesting that these organelles may be attractive targets for the development of novel therapeutics. Transposon mutagenesis studies have identified the pilMNOPQ genes as being essential for T4P assembly and twitching motility. This study investigates the roles of the type II membrane proteins PilNO. Methods: Sucrose gradient fractionation was used to determine the subcellular localization of each PilMNOP protein in the P. aeruginosa PAO1 strain. Vectors to express the periplasmic domains of PilNO both individually and together have been generated and the purified proteins biochemically characterized. The structure of the periplasmic domain of PilO was solved using X-ray crystallography. Results: Our in vivo studies indicate that PilMNOP proteins, that have been shown to exert a mutual stabilization effect on each other, co-localize to the inner membrane. While in vitro PilO forms soluble homodimers, expression of PilN results in mostly insoluble protein. Co-expression of PilN/O improves the stability of PilN and produces PilNO heterodimers that can be readily purified. The 2.2Å structure of PilO revealed two domains, an N-terminal coiled-coil and a C-terminal ferredoxin-like domain. This structure was used to generate a homology model of PilN and PilNO heterodimer. The PilNO model has been tested by site directed mutagenesis of residues that reside on the putative dimerization interface. Conclusion: Our data suggest that formation of a PilMNOP sub-complex is required for T4P assembly and we hypothesize that the stable PilNO heterodimer we have characterized anchors this complex in the inner membrane.


Virulence strategy of Pseudomonas aeruginosa in acute infections on the basis of secretion systemsGiovanna Scalet1, Dinesh Diraviam Sriramulu2 and Olivier Jousson2

1Pathology Department-Microbiology Section, University of Verona, Strada Le Grazie 8 37134 Verona-Italy, 2Centre for Integrative Biology, University of Trento, Via delle Regole 101, Mattarello (TN) 38060, Italy

P. aeruginosa possesses an arsenal of virulence factors and displays a remarkable range of virulence, from non-virulent to highly virulent broad spectrum strains. Protein secretion systems are among virulence determinants that mediate interactions between bacteria and eukaryotic host cells, and contribute to the severity of infections. There are 6 secretion systems known in gram negative bacteria. Whereas some secretion systems of P. aeruginosa have been intensively studied in a small number of reference strains, there is, with the exception of type III secretion system, a lack of data regarding their distribution and relevance in clinical strains. Type I, II, III, IV, V and VI have been detected in P. aeruginosa. This project focused on the study of the protein secretion systems in clinical isolates of P. aeruginosa involved in acute infections. Our collection consists of 113 P. aeruginosa strains isolated from 69 patients: 65% from intensive care unit, 29% from hematology and 6% from other clinical departments. 55% of strains were isolated from patients with bacteremia while the remaining isolates originated from other acute cases. Genotyping using rep-PCR allowed us to classify 76 strains in 13 clusters and the remaining 37 strains showed unique rep-PCR profiles. PFGE profiling was carried out for representative strain

98


from each cluster and also for strains with unique rep-PCR profiles. Genotyping revealed the presence of a predominant clone (rep-PCR clone A) isolated from both Hematology and Intensive Care Unit demonstrating that it has been cross-transmitted in the nosocomial environment. Antibiotic resistance-profiling showed that 70% of the strains were resistant to carbapenems, 60% to cephalosporins, 57% to aminoglycosides and 53% were multiresistant. Profiling of genes involved in secretion systems by PCR showed variable distribution of effectors associated with type I-V secretion systems in these strains. Interestingly, all P. aeruginosa strains in our collection carried genes that encode effectors associated with type VI secretion system.


Survey of the role of peptidoglycan remodeling enzymes in facilitating assembly and function of cell-envelope spanning structures in Pseudomonas aeruginosa PAO1Edie M Scheurwater1 and Lori L Burrows1

1McMaster University, Hamilton, Canada

Spanning the cell-envelope of Pseudomonas aeruginosa are a variety of virulence-associated, surface-exposed, multiprotein complexes, including flagella, type IV pili, secretion systems, and efflux pumps, that must assemble in such a way as to avoid disruption of the structural integrity of the peptidoglycan (PG) layer. It has been suggested that this safe assembly occurs via controlled localized remodeling of the PG layer by specific enzymes, though this has yet to be systematically studied in any organism. P. aeruginosa encodes more than 25 different enzymes predicted to act on the PG layer, many with seemingly redundant activites, but whose functional roles need to be clarified. Previous work in our lab showed that perturbations of PG biosynthesis/remodeling in P. aeruginosa impaired important virulence related phenotypes. To understand how loss of specific PG enzymes impacts phenotypes associated with virulence in P. aeruginosa, we initiated a comprehensive phenotypic survey of a PAO1 PG transposon (Tn) mutant panel (49 strains representing 25 ORFs) to identify mutants with altered antibiotic susceptibility and aberrant swimming, twitching, secretion, or static biofilm formation. Mutants in 7 different PG enzymes showed altered sensitivity to one or more antibiotics tested including Tn mutants of PBP4, MltF, and SltB1 which showed increased resistance to carbenicillin and piperacillin, norfloxacin, and piperacillin, respectively. Mutations in PBP1a showed a decrease in swimming motility, but no loss in amount of surface flagella, a phenotype also present in an unmarked PBP1a mutant that could be complemented. A number of mutant strains had atypical twitching, including PBP3a, MltF, PBP6, AmiB, and ExeA. These strains also had atypical type II secretion, suggesting that these structurally related complexes may share a similar association with the PG layer. Follow-up studies showed a decrease in the amount of surface pili produced by the PBP3a and AmiB Tn mutants. Interestingly, the AmiB and ExeA Tn mutants also produced less biofilm compared to wildtype. Although P. aeruginosa produces PG enzymes with seemingly redundant activities, we show that some may have specific roles in the assembly of multiprotein structures involved with virulence. Further studies to relate changes in PG structure with effects on the function of multiprotein complexes are underway.


SprS of Pseudomonas aeruginosa: a novel autotransporter protease influences the expression of other proteasesStephanie Serci1, Frank Rosenau1 and Susanne Wilhelm1

1Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf at Forschungzentrum Juelich, Germany

In many Gram-negative organisms autotransporter proteins with a diverse array of N-terminal functional domains have been reported (e.g., Bordetella pertussis, Heamophilus influenza and Neisseria meningitidis). These proteins typically reveal virulence-associated functions such as adhesion, serum resistance, cytotoxicity and proteolysis. Here we introduce a novel autotransporter of P. aeruginosa which we have called SprS. The deduced amino acid sequence showed homology to several subtilisin-like autotransporter proteins of pathogenic bacteria like SphB1 from Bordetella pertussis and NalP from Neisseria meningitides. These proteins are known to be involved in the proteolytic processing of secreted proteins like the precursor FHA. Since P. aeruginosa PAO1 also possesses FHA homologues, we compared the agglutination activity of the wild type to the sprS-negative strain, in order to see whether SprS might be involved in FHA processing. Previous studies of the secretome have shown that the sprS-negative strain produces less virulence associated proteases than the wild type. Therefore we have been analyzing the expression of sprS and other proteases in the sprS-negative strain. We have shown that the expression of other protease genes is influenced by SprS. As a consequence this is an indication of coordinated regulation of proteases in a network like manner.


RNase E is required for expression of the type III secretion system in P. aeruginosa Josh S. Sharp1, Charles Stopford2, Arne Rietsch 2 and Simon L. Dove1

1Children's Hospital Boston - Harvard Medical School, 2Case Western University, USA

The type III secretion system of P. aeruginosa alters host cell physiology by delivering toxic effectors, is essential for acute infection, and is highly regulated. Using a ClpXP protease-based depletion system we show that ribonuclease E (RNase E), an essential endoribonuclease, is required for induction of the type III secretion system. In particular we show that depletion of RNase E prevents induction of type III secretion gene expression under conditions of low calcium. Furthermore, DNA microarray analyses indicate that when RNase E is depleted, expression of genes involved in type III secretion are significantly down-regulated, while the expression of those genes involved in biofilm formation, type VI secretion, and hydrogen cyanide synthesis are up-regulated. Thus, RNase E appears to exert global effects on the expression of virulence genes. Finally, we present evidence that RNase E does not mediate its effects through RsmY or RsmZ, two small RNAs which have been shown previously to reciprocally regulate the expression of the type III and type VI secretion genes.


The lipoprotein HxcQ: a new class of secretin with an intramolecular pilotin Véronique Viarre1, Eric Cascales1, Geneviève Ball1, Gérard Michel1, Alain Filloux2 and Romé Voulhoux1

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1Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), UPR9027, IFR88/CNRS, 31 Ch. J. Aiguier, 13402 Marseille Cedex 20, France, 2Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

Bacterial secretins are unusual outer membrane (OM) proteins involved in the transport of large structures such as pili, needle complexes, phages or folded proteins across the OM. They form large, donuts-shaped pores in the OM which require a specific and well adapted biogenesis pathway. In many envelope transport machines, a small OM lipoprotein is often associated with the OM pore. This is also the case for secretins and their cognate pilotins which play an important role in the correct transport of the secretin to its final destination. In the present study, we report that the secretin of the second type II secretion systems, HxcQ, is itself a lipoprotein. We clearly demonstrate that the N-terminal lipid anchor of the HxcQ liposecretin fully fulfils the pilotin function. First, a recombinant non lipidated HxcQ secretin (HxcQnl) behaves similarly to a classical secretin in absence of its cognate pilotin (multimers of HxcQnl inserted into the inner membrane: pilotin minus phenotype). Second, the reconstitution of a classical extramolecular trans pilotin assistance retarget HxcQnl to the OM. Together our data demonstrate the existence of an intramolecular pilotin within the HxcQ liposecretin. Besides its novelty, this finding suggests that the major function of a pilotin is to address its cognate secretin to the Lol general lipoprotein transport system for its transport to the OM.


Microarray-based detection of multidrug efflux systems in the plant pathogen Pseudomonas syringaeHelge Weingart1, Patricia Wecker2, Andreas Ellrott2 and Matthias Ullrich1

1Jacobs University Bremen, 2MPI für Marine Mikrobiologie, Bremen, Germany

Plant-pathogenic bacteria are continually exposed to deleterious chemicals naturally occurring in their environment such as antimicrobial plant metabolites and toxins produced by other microorganisms. Bacteria have developed various ways to resist the toxic effects of antimicrobial compounds. Extrusion of toxic agents from cells by multidrug efflux (MDE) is one of these mechanisms. This project aims to identify and characterize MDE pumps in the plant pathogen Pseudomonas syringae pv. tomato DC3000, the causal agent of bacterial speck on tomato and Arabidopsis.The genome sequence of P. s. pv. tomato DC3000 was used to develop a gene-specific oligonucleotide microarray containing open reading frames predicted as putative multidrug pumps. The 83 oligonucleotides were selected from genes sharing sequence similarity with established MDE transporters plus efflux systems with unknown function and no similarity to characterized transporters. The microarray was used to identify transporters that are expressed during growth of P. s. pv. tomato DC3000 in induction medium supplemented with plant extracts and antimicrobial plant metabolites, respectively. To learn more about the natural functions of these transporters and their role during pathogenesis, we will analyze the expression of these efflux pumps during infection of the host plants.


Export and periplasmic maturation of the siderophore pyoverdine in Pseudomonas aeruginosaEmilie Yeterian1, Lois Martin2, Iain Lamont2 and Isabelle Schalk1

1IREBS/CNRS, University of Strasbourg, 2University of Otago, PO Box 56, Dunedin, New Zealand

Pyoverdines (PVDs), the main siderophores produced by fluorescent Pseudomonads under iron-limiting growth conditions, are able to chelate Fe3+ with a strong affinity. All pyoverdines contain a peptide moiety and an identical chromophore. The biosynthesis of the PVD backbone involves cytoplasmic non-ribosomal peptide synthetases (NRPSs) and different enzymes. Nothing about export and late step of maturation of PVDI, the PVD produced by Pseudomonas aeruginosa, is elucided. Using the fluorescent properties of PVDI, we show by microscopy fluorescence and fractionation that large amounts of PVDI were stored in the periplasm, but no PVDI was present in the cytoplasm of this bacterium. Mutation of an export ABC transporter in the inner membrane affected the PVDI production and no more fluorescence was observed in the periplasm. The function of this transporter is apparently to transport a PVDI precursor with a non-formed chromophore accros the inner membrane. Mutation of the periplasmic PvdN, PvdO and PvdP proteins affected PVDI production and no more fluorescence was observed in the periplasm of the bacteria. These enzymes must be involved in either the cyclisation of the chromophore or a step preceeding it. The PVDI synthesis pathway will be discussed in the light of these different new data.

Keynote Lectures II

K-03 Topic: Translational Research for Biotechnology, Medicine and Agriculture

The challenges of Pseudomonas vaccine development: from here to immunityJoanna B. Goldberg1


Pseudomonas aeruginosa is an opportunistic pathogen that causes life-threatening infections in compromised patients. It is a major common cause of ventilator-associated pneumonia, and acute infections following cancer chemotherapy, burns, and eye injury. It is also responsible for most of the morbidity and mortality in cystic fibrosis (CF) patients due to chronic lung infections. Intrinsic and acquired resistance to antibiotics as well as its biofilm mode of growth makes infections caused by P. aeruginosa particularly difficult to treat. Vaccination has been considered an appropriate approach to prevent or limit infections caused by this bacterium. For the most part, the candidates that have been tested have been prominent and immunodominant antigens expressed by P. aeruginosa. However, to date, no immunotherapeutic agents have progressed beyond clinical trials. The release of the first P. aeruginosa genome sequence and that of additional strains, as well as post-genomic technologies that have followed, has renewed interest in identifying viable vaccine candidates. However, vaccinology remains an empirical science. The general approach is to target antigens that are surface exposed. In addition, suitable candidates must be expressed by the infecting organism. This is particularly critical when considering P. aeruginosa as the phenotypes of strains causing acute infections are distinct from those responsible for chronic lung infections in CF. While the most obvious difference is that strains from chronic infections have a mucoid phenotype due to the overproduction of the exopolysaccharide alginate, numerous other differences have been recognized by phenotypic, transcriptome, and proteome analyses. Added challenges in testing potential vaccines include safety issues, defining routes

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of delivery to induce an appropriate an immune response, and choosing an animal model that accurately mimics the infectious process. For human trials, there is the additional consideration of selecting an appropriate endpoint to define efficacy, and issues related to low infection rates among placebo control groups. Vaccine candidates that are currently being investigated for active immunization include purified polysaccharides and proteins, attenuated P. aeruginosa strains, antigens expressed in heterologous systems, and polysaccharide-protein conjugates. In addition, passive immunotherapies are being developed to combat this pathogen after it has initiated an infection.

K-04 Topic: Genomics and Metagenomics

Antibiotic resistance, epithelial cell interactions and swarming motility in Pseudomonas as complex adaptationsRobert Hancock1

1University of British Columbia, Canada

Pseudomonas aeruginosa has the ability to adapt its lifestyle to many different circumstances using a wide variety of genes, a process we have termed complex adaptation; indeed its prominence as an opportunistic pathogen seems to reflect this. The circumstances that will be discussed here are antibiotic treatment, epithelial cell interactions and swarming motility. Two types of studies lead to the conclusion that these are complex adaptations. Treatment with subinhibitory concentrations of antibiotics, such as ciprofloxacin and cationic antimicrobial peptides, lead to the up- and down- regulation of dozens to hundreds of genes. Conversely screening of comprehensive knockout libraries for resistance to antibiotics can lead to dozens of genes that influence susceptibility in the case of ciprofloxacin and gentamicin, and more modest numbers in the case of beta-lactams and peptides. Similar types of results were observed for epithelial cell interaction and swarming motility. The latter case, distinct from flagellum-mediated swimming in aqueous environments, and type IV pilus-mediated twitching on solid surfaces/interfaces, involves dendritic-like swarming on semi-solid media (0.5-0.7% agar) containing amino acids as a nitrogen source (conditions thought to mimic the cystic fibrosis lung). Microarray analysis of bacteria at the leading edge of a swarm zone, compared to bacteria growing in identical medium under swimming conditions revealed 417 genes that were significantly up- (309 genes) or down- (108 genes) regulated by more than 2-fold, influencing virulence and antibiotic resistance. Eighteen were predicted or known regulators with 2- to 11-fold altered expression, indicating a complex regulatory network. Conversely, 226 out of the 5600 P. aeruginosa PA14 transposon library mutants were verified as having alterations in swarming motility and 33 were regulators. Interestingly there was limited overlap between the genes necessary for swarming and those dysregulated under swarming conditions. These results will be discussed with respect to three major aspects (1) the overlap between the three adaptations with special reference to particular genes and how these impact on virulence and antibiotic resistance, (2) initial approaches to defining regulatory hierarchy, and (3) establishment of bioinformatics tools to understand how these complex biological systems are integrated (PaintDB).

101


102


Authors

103


A

Aaron, Shawn

P-17

Abdou, Laetitia

P-35

Åberg, Anna

P-113

Abraham, Wolf-Rainer

P-93; P-98

Ackerley, David F.

P-36; P-56

Adams, Claire

PO-153; P-169; P-176; P-211

Adams, Thorsten

P-69

Adonizio, Allison

P-134

Agathos, Spiros N.

P-25

Ahuja, Ekta

PO-30

Albers, Alexander

P-138

Allada, Gopal

104


Cho, You-Hee

P-39; P-47; P-51; P-78;

P-116; P-166

Choi, Kyoung-Hee

P-39; P-47; P-160

Choi, Yu Sang

PO-151

Christie-Oleza, Joseph Alexander P-5

Chugani, Sudha

P-117

Chung, In-Young

P-39; P-47

Chung, Jade C.S.

PO-156; P-161

Clardy, Jon

P-134

Clatworthy, Anne

P-149

Codina, Juan C.

P-97

Connell, Jodi

P-146

Cool, Robbert

P-167; P-175

Coorevits, An

P-79

P-134

de Bentzmann, Sophie

O-18; P-96; P-196

De Block, Jan

P-79

de Eugenio, Laura

P-86

De Jonghe, Valerie

P-79

de Lorenzo, Victor

K-1; P-16; PO-33; PO-82;P-87

De Mot, René

P-52; P-105

de Vicente, Antonio

P-6; P-97

De Vos, Paul

P-79

De Winde, Han

PO-75

Dean, Charles R.

P-172

Degrassi, Giuliano

P-128

del Peso Santos, Teresa

P-118

Dessen, Andrea

105


Gallegos, Mari-Trini

P-182

Garbe, Julia

P-199

García, Jose Luis

P-86

García-Jaramillo, Manuel

P-212

Geffers, Robert

P-9; P-128

Gendrin, Claire Sophie

P-200

Gesell-Salazar, Manuela

PO-81

Gil, Concha

P-55

Gilbert, Kerrigan

PO-92

Giltner, Carmen L.

P-11

Giraud, Caroline

P-96

Givskov, Michael O-6

Gjermansen, Morten

O-13

Glik, Justyna

P-157

PO-74

Guazzaroni, María Eugenia

P-5; P-44; P-62

Guittet, Eric

PO-155

Gulbins, Erich

PO-73

Gupta, Rashmi

P-126

Gutiérrez-Barranquero, Jose A.

P-6; P-97

H

Haas, Dieter

O-14; PO-32; P-35; PO-110;

P-137

Habash, Marc

P-11

Hachani, Abderrahman

PO-31; P-192

Haddad, Ahmed Mahmoud

P-127

Hageluecken, Gregor

106


Kato, Tomohisa

P-124

Katsuki, Junichi

P-66

Kawasaki, Aika

P-65

Kawecki, Marek

P-157

Keiski, Carrie-Lynn

P-202

Kessleer, Efrat

P-194

Khaira, Bhavjinder

P-28

Kim, JungEun

P-78

Kim, Seol-Hee

P-51; P-166

Kim, Soo Kyoung

PO-151

Kirkelund Hansen, Susse

O-9; P-12

Kirov, Sylvia

P-184

Kitamura, Chitoshi

P-65

Kivisaar, Maia

P-206

Krell, Tino

P-62

Kremling, Andreas

P-87; P-90

Krull, Rainer

P-180

Ku, Shao-Yang

P-205

Kuehne, Sarah Anna

P-140

Kühn, Christian

P-93

Kukavica-Ibrulj, Irena

P-14; P-38

Kuroki, Miho

P-15

Kwon, Taejoon

P-50

L

Lacal, Jesús

P-62

Lalucat, Jorge P-5

Lam, Joseph S.

P-199; P-207

107


McManus, Heather Rose

P-210

Mentel, Matthias

PO-30

Merighi, Massimo

O-14

Messina, Marco

PO-111; P-140; P-144

Mettrick, Karla

P-132

Mi, Xiaoyuan

P-178

Michel, Gérard

P-220

Mikkelsen, Helga

PO-156

Milani, Andrea P-4

Miller, Helen

P-174

Minagawa, Shu

P-124

Miyairi, Shinichi

P-124

Miyakoshi, Masatoshi

P-19

Moeker, Nina

P-172

Molin, Søren

Murphy, Timothy F.

O-15; P-179

Müsken, Mathias

P-20; P-63

N

Nadal Jimenez, Pol

P-167; P-175; P-177

Nagata, Yuji

P-19

Naito, Kunihiko

P-71

Nassafi, Khaled Al

PO-1

Navazo, Ana

P-76; P-101; PO-112

Nechitaylo, Taras P-7

Neculai, Mirela

P-202

Negoro, Seiji

P-65

Neu, Thomas

P-98

Neugebauer, Jens P-8

Newman, Dianne

O-4; PO-150

Nielsen, Jens

108


Rajakumar, Kumar

P-164

Rakhimova, Elza

P-57; P-179

Ramalingam, Bharathi

P-134

Ramos, Juan Luis

P-62; PO-109

Ramotar, Karam

P-17

Rampioni, Giordano

P-121; P-139

Randel, Nadine

P-60

Rau Holm, Martin O-9

Redondo-Nieto, Miguel

P-76; P-101; PO-112

Reen, Jerry

P-22; P-84

Rehel, Karine

P-145

Rehm, Bernd HA

P-46; PO-186

Reimmann, Cornelia

PO-34; P-137; P-148; PO-185

Remminghorst, Uwe

P-37

Ruijssenaars, Harald J

PO-75

S

Saito, Shizuka

PO-149

Salacha, Richard

P-214

Sampaleanu, Liliana

P-190; P-205; P-215

Sanschagrin, François

P-14

Santero, Eduardo

P-114; P-212

Sarkisova, Svetlana

P-104

Sauer, Marie

P-180

Saumaa, Signe

PO-2

Sawada, Shinji

P-124

Scalet, Giovanna

P-216

Scarff, Jennifer M.

109


Tsuda, Masataka

P-19

Tümmler, Burkhard

P-8; P-43; P-49; P-57;

P-69; P-90; P-95; P- 157

P-179; P-183

Turnbull, Lynne

P-171

Turner, Keith H.

P-181

Tye, Laura

P-144

U

Ullrich, Matthias

P-221

Ulmer, Christoph

P-43; P-90

Ulrich, Martina

O-14

Umeda, Takashi

P-66

Urbanke, Claus

P-41; P-69

Usami, Yusuke

Ventre, Isabelle

PO-31

Venturi, Vittorio

P-128; P-133

Veron, Wilfried

P-145

Verove, Julien

P-193

Vettoretti, Lucie

P-208

Vial, Ludovic

P-125

Viarre, Véronique

P-220

Vieites, Jose M. P-5

Villaseca, Christian

P-134

Vinckx, Tiffany

P-67

Völker, Uwe

PO-81; P-90

von Specht, Bernd-Ulrich

P-77

Voulhoux, Romé

P-197; P-206; P-214; P-220

W

110


111

Documents

Pseudomonas 2009, XII International Conferencegenomics1.mh-hannover.de/pseudomonas2009/Startseite/Includes/img… · “Pseudomonas: Pathogenicity and ... Lecture Hall F August 13-17,