Monday 20 August PS09 – Pathways and Physiologyllebbe/Abstracts/ISME 14 ABSTRACT BOOK/Mo… · PS09 – Pathways and Physiology . 453A Biodegradation of C. 7. and C. 8. iso-alkanes

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1 PS09 – Pathways and Physiology

PS09 – Pathways and Physiology

453A Biodegradation of C7 and C8 iso-alkanes under methanogenic and sulfate-reducing conditions Nidal Abu Laban*, Kathy Semple, Rozlyn Young, Anh Dao, Julia Foght University of Alberta/Department Biological Sciences, Canada

Oil sands tailings ponds in northeastern Alberta, Canada contain unextracted bitumen as well as residual naphtha used during extraction of bitumen from oil sands ores. n-Alkanes and some monoaromatics in naphtha are biodegraded to methane by indigenous microbes in the tailings ponds. However, the biodegradability of other naphtha components such as iso- and cyclo-alkanes is unstudied. Here we document anaerobic degradation of C7 and C8 iso-alkanes (3-methylhexane and 4-methylheptane) by enrichment cultures derived from oil sands tailings incubated under methanogenic or sulfate-reducing conditions. Phylogenetic analysis of the cultures using 454-pyrotag sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed predominance (83.5%) of 16S rRNA genes affiliated with Clostridiales, particularly Cryptanaerobacter (T-RF 172 bp) and Proteiniclasticum (T-RF 515 bp) under methanogenic conditions, whereas Deltaproteobacteria, particularly Desulfobacteriales/Desulfobulbaceae (T-RF 157 bp), increased in abundance (to 61.9%) with sulfate as the electron acceptor. To investigate possible activation mechanisms for these iso-alkanes, we used GC-MS to analyze trimethylsilyl-derivatized metabolites extracted from the enrichments. In both methanogenic and sulfate-reducing cultures, we found derivatives with mass spectra consistent with putative succinyl-C7 and -C8 metabolites but with retention times different from n-alkylsuccinates (for example C8 n-octylsuccinate). This suggests the production of methyl-branched alkylsuccinate metabolites during anaerobic degradation of 3-methylhexane and 4-methylheptane. This inference is supported by amplification of putative assA (alkylsuccinate synthase)-like gene sequences possibly encoding glycyl radical enzymes that catalyze addition of iso-alkanes to fumarate. Thus we propose that members of the Clostridiales and Desulfobacteriales are involved in the initial activation of iso-alkanes via addition to fumarate under methanogenic and sulfate reducing conditions.

454A Are microorganisms involved in freshwater humic matter degradation? A metagenomics approach Martin Allgaier*1, Ivette Salka2, Hans-Peter Grossart2 1Berlin Center for Genomics in Biodiversity Research, Germany, 2Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany

Humic substances (HS) are a complex and heterogeneous mixture of polymers formed in soils, sediments and natural waters by physico- and biochemical transformation of plant biomass (humification). They are primarily composed of polysaccharides, phenolic compounds (e.g. lignin, tannins), amino acids, proteins and various other compounds. HS represent an important carbon source in freshwater ecosystems accounting for up to 80% of the total dissolved organic carbon pool. Based on current structural concepts it is assumed that HS cannot be degraded by simple enzymatic reactions, but requires a large number of different enzymes and a complex interplay of different organisms.

To study the role of microorganisms in HS degradation in freshwater we performed a metagenomic study of aerobic and anaerobic microbial communities in dystrophic Lake Grosse Fuchskuhle, Germany. A total of 213 Mbp of sequence data were generated (75 Mbp for aerobic and 135 Mbp for anaerobic communities) and analyzed towards their capabilities to degrade HS. Both communities showed a substantial fraction (up to 10 %) of genes involved in carbohydrate metabolism including cellulases and hemicellulases. Thereby endo-active enzymes (e.g. endoglucanases) were only found in anaerobic communities. We also identified several enzymes potentially involved in direct degradation of HS (e.g. phenol hydroxylases or catechol dioxygenases). The data sets will be further analyzed to obtain a more detailed overview on the microorganisms involved in HS transformation in the lake and to resolve their ecological role in these habitats. Our preliminary data suggest that microbial degradation greatly differs between oxic and anoxic environments and that stratification and mixing patterns have a severe effect on microbial HS degradation.

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455A Microbial influences on arsenic release from Japanese paddy soils Seigo Amachi*1, Keitaro Kudo1, Eri Honma1, Diantao Dong1, Tomoyuki Makino2, Noriko Yamaguchi2 1Chiba University, Japan, 2National Institute for Agro-Environmental Sciences, Japan

Predominant arsenic species in soil environment are arsenate (As(V)) and arsenite (As(III)). Under oxic conditions, As(V) is strongly sorbed on Fe mineral phases such as ferric (hydr)oxide. Under reducing conditions, on the other hand, ferric (hydr)oxide undergoes reductive dissolution, and arsenic sorbed on mineral phases is released mainly as As(III). Metal-reducing bacteria such as Geobacter spp. may play key roles in the process of arsenic release, either through reduction and dissolution of arsenic-bearing Fe minerals, or through a direct reduction of sorbed As(V). Since arsenic release occurs in flooded paddy soils, rice is a major source of dietary intake of inorganic arsenic in the Japanese population. In this study, we determined microbial influences on arsenic release from Japanese paddy soils by using culture-dependent and -independent approaches.

Paddy soil (20 g dry weight) containing approximately 40 mg kg-1 of total arsenic was mixed with 60 g of dionized water in a 120-mL glass vial. After N2 gas flushing, the vial was sealed and incubated for 60 to 100 days. At various time intervals, the slurry was centrifuged and Eh, pH and arsenic speciation (HPLC-ICP/MS) in the supernatant were analyzed. For isolation of As(V)-reducing bacteria, soils were anaerobically enriched with acetate and As(V). Genes encoding arsenate respiratory reductase (arrA) were amplified from DNA extracted from pure cultures, enrichment cultures and soils by two sets of degenerate primers AS1F/1R and AS2F/1R (Song et al., 2009). Arsenic speciation in soil solid phase was determined by K-edge X-ray absorption near-edge structure (XANES) spectra.

Anaerobic incubation of paddy soils resulted in the drop of Eh, rise in pH, and release of As(III) in the liquid phase. At the end of incubation, As(III) concentrations reached to 200 to 400 µg L-1. When the soils were autoclaved, little As(III) was released. Three As(V)-reducing bacterial strains OR-1, SHIMR-1 and PSR-1 were successfully isolated from enrichment cultures. 16S rRNA gene analyses showed that these strains were closely related with Geobacter pelophilus (98%), Desulfitobacterium metallireducens (95%) and Anaeromyxobacter dehalogenans (99%), respectively. All of these strains could grow with As(V) or Fe(III) as the sole electron acceptor. arrA was amplified from genomic DNA of strain OR-1, and its amino acid sequence (ArrA) showed 86 to 90% similarities with those found on the genomes of G. lovleyi SZ and G. uraniireducens Rf4. Inoculation of the newly isolated pure cultures into sterile paddy soils could restore As(III) release (70 to 140 µg L-1). As(III) concentration in the liquid phase showed a significant correlation (R2=0.89) with the proportion of As(III) in the solid phase, suggesting that As(V) is reduced on the soil solid phase. Phylogenetic analysis of putative arrA genes of paddy soils revealed that about half of arrA genes are closely related with Geobacter spp. These results suggest that Geobacter may play significant roles in arsenic release from Japanese paddy soils.

456A Caspase homologs in prokaryotic genomes and metagenomes Johannes Asplund Samuelsson*1, John Larsson1, Chris L Dupont2, Shibu Yooseph2, Johannes Goll2, Mathangi Thiagarajan2, Karolina Ininbergs1, Narin Celepli1, Björn Brindefalk1, Birgitta Bergman1 1Stockholm University, Sweden, 2The J. Craig Venter Institute, United States

Caspases inititate and execute apoptosis, a metazoan programmed cell death process. Homologs of these proteases, here referred to as metacaspases, are known to exist in prokaryotes, and in the few instances they have been studied, they have been implicated in programmed cell death. We applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial/archaeal genomes and in a metagenomic dataset obtained from the Baltic Sea (part of the 2009 Global Ocean Sampling Expedition). Putative metacaspases were identified in 267 out of a total of 1463 analyzed genomes and occur in most sequenced prokaryotic groups, including the Euryarchaeota. Metacaspases are particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms. In contrast, Bacillus subtilis and Escherichia coli, able to undergo genetically regulated autolysis, lack metacaspases. The absence of metacaspases in early-branching bacteria and most archaeal phyla suggests a late emergence of caspases, followed by horizontal transfer to archaea and eukaryotes. Analyzing metacaspase domain architectures (Pfam)

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revealed a high diversity, implying roles in programmed cell death, but also for instance in signaling, various enzymatic activities and protein modification. Additionally, unique metacaspase domain architectures identified in the Baltic Sea metagenome implies a large pool of novel metacaspase sequences among prokaryotes. The greatest metacaspase-frequency in the Baltic Sea metagenome was found in Deltaproteobacteria, Nitrospirae and Epsilonproteobacteria, which suggests significant differences between natural populations and sequenced cultured microorganisms. Furthermore, the larger Baltic Sea bacteria carry a higher proportion of metacaspases in their genomes, strengthening the notion that metacaspases relate to more complex bacteria. Evidently, metacaspases represent a functionally diverse and widely distributed enzyme among prokaryotes.

457A Physiological characteristics of marine anammox bacteria enriched from sea sediments, Hiroshima, Japan Takanori Awata*1, Tomonori Kindaichi1, Noriatsu Ozaki1, Akiyoshi Ohashi1, Mamoru Oshiki2, Satoshi Okabe2 1Hiroshima University, Japan, 2Hokkaido University, Japan

Anaerobic ammonium oxidation (anammox) is a microbial reaction, in which NH4+ is oxidized to N2 gas

with NO2- as electron acceptor. Anammox reaction is mediated by the members in the bacterial order

'Brocadiales' deeply branched in the phylum Planctomycetes. At least five candidate genera has been tentatively proposed in this taxon as follows; 'Candidatus Brocadia', 'Candidatus Kuenenia', 'Candidatus Scalindua', 'Candidatus Jettenia', and 'Candidatus Anammoxoglobus'. Niche speciation among these anammox bacteria in natural and man-made ecosystems has been investigated by 16S rRNA gene-based clone library or T-RFLP analysis, which revealed that the members of 'Candidatus Scalindua' are marine anammox species. Although the importance of anammox reaction in the marine nitrogen cycle has been well documented, the physiology of marine anammox species is largely unknown because the physiological characteristics have not been investigated with marine anammox species and the past studies mainly focused on the freshwater anammox species (i.e. 'Candidatus Brocadia' and 'Candidatus Kuenenia'). To obtain more comprehensive knowledge about the ecology and the niche speciation of marine anammox species, the study investigating their physiological characteristics is inevitably essential.

The authors previously succeeded to enrich marine anammox bacteria from the sea sediment in Hiroshima, Japan, and the objectives of this study were to investigate their phylogenetic affiliation and physiological characteristics. Phylogenetic affiliation of the marine anammox bacteria was investigated by determining the nucleotide sequences of 16S rRNA gene. Subsequently, we designed a specific oligonuleotide probes specific and used in the fluorescent in-situ hybridization analysis to determine the abundance of the marine anammox bacteria in the enriched biomass. Physiological characteristics were investigated in the vial test where the biomass of the marine anammox bacteria was anaerobically incubated at the different salinity concentration and the isotopomer analysis for N2 gas was performed to determine their anammox activity. Our results are beneficial to understand the niche speciation between the freshwater and marine anammox species.

458A Novel findings on degradation metabolic pathways of endocrine disrupters, persistent organic pollutants and pharmaceuticals by ligninolytic fungi Tomas Cajthaml*1, Zdena Kresinova2, Monika Cvancarova2, Alena Filipova2, Stefano Covino2, Tatiana Stella3, Monika Moeder4 1Institute of Microbiology, Academy of Sciences , Czech Republic, 2Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Czech Republic, 3Università degli Studi della Tuscia, Italy, 4UFZ-Centre for Environmental Research Leipzig-Halle, Germany

Ligninolytic (white rot) fungi possess a unique degrading enzyme apparatus and it has been documented that they are able to biodegrade a wide range of organic pollutants including xenoestrogens, aromatic hydrocarbons, polychlorinated biphenyls and others. This is mainly due to their ability to excrete low-substrate specific extracellular enzymes together with detectable activity of cytochrome P-450 monooxigenase. Despite indisputable biotechnological prospects, limited information is available about biotransformation products of these pollutants and physiology of the degradation is not well understood. In this contribution, we present novel metabolites of various types of recalcitrant aromatic pollutants that are effectively degraded by ligninolytic fungi. Moreover, we tried to identify also participation of different enzyme machineries in the degradations using isolated

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enzymes or sub-cellular fractions. The relevant organic pollutants under our study were: endocrine disrupters - including bisphenol A and 17α-ethinylestradiol (EE2); persistent organic pollutants including polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCBs); pharmaceuticals - including diclofenac, flumequin, ofloxacin and others.

Using detailed analysis, we found that e.g. the fungi degrade PCB congeners effectively (up to 95%) and characterization of the degradation products revealed that the fungi transform PCBs via hydroxylated and methoxylated derivatives to chlorobenzoic acids that are further transformed via a reductive pathway. This was also demonstrated for variously chlorinated biphenyls. Chlorobenzoic acids represent crucial recalcitrant bacterial PCB metabolites; however, as opposed to bacteria, the fungi can efficiently perform the transformation further to form less toxic products as we proved.

Another important and relevant pollutant (EE2) was also transformed efficiently and the data of the investigations suggest the occurrence of asynergistic action of several metabolic mechanisms. These could be attributed to: intracellular degradation via microsomal enzymes, mycelium associated laccase-like activity, extracellular degradation via manganese-dependent peroxidase and extracellular laccase. Using numerous in vitro experiments, the contribution of each enzyme activity to pollutant degradation was assessed and the ability to remove the associated estrogenic activity was evaluated.

The results of our studies clearly show that fungi use various mechanisms for the transformation of aromatic pollutants, including cytochrome P-450, aryl-aldehyde dehydrogenases, aryl-alcohol dehydrogenases, methyltransferases and probably other enzymatic systems acting together with the extracellular ligninolytic machinery. However, contrary to generally accepted theories, the ligninolytic system is not always responsible for the initial attack of the pollutants.

459A Genome characterization of Pelosinus fermentans JBW45 isolated during in situ stimulation for Cr(VI) reduction Laura Camilleri*1, Kara De Leon1, Mary Young1, Steven Brown2, Jeffrey Skerker3, Morgan Price3, Adam Deutschbauer3, Adam Arkin3, Matthew Fields1 1Montana State University, United States, 2Oak Ridge National Laboratory, United States, 3Lawrence Berkeley National Laboratory, United States

The Hanford Nuclear Reservation 100-H site (Washington, USA) is a Chromium (VI) contaminated site designated as a field study site for bioremediation by the Department of Energy. In 2008, a Hydrogen Release Compound® (HRC®) was injected into the well water in order to stimulate microbial chromium reduction. After 24 hours, samples from the well water were collected to enrich for sulfate-reducing populations with lactate and sulfate using LS4D media. Sulfate-reducing bacteria were specifically targeted due to the ability to reduce heavy metals (e.g, chromium). The cultures in LS4D were diluted to extinction and single colonies were isolated on LS4D plates. During the isolation process, sulfide production was initially observed but declined upon subsequent transfers. Enrichment cultures were predominated by Pelosinus populations, and the isolation of Pelosinus fermentans JBW45 could indicate that HRC® stimulation for chromium reduction might also stimulate lactate-oxidizing populations other than metal-reducing bacteria. These results coincided with the observation that the number of groundwater Pelosinus sequences increased after in situ stimulation. The draft genome sequence for P. fermentans JBW45 resulted in 98 contigs greater than 500 bp. The draft genome sequence was annotated at Oak Ridge National Laboratory using an automated annotation pipeline, based on the Prodigal gene prediction algorithm. A total of 4765 candidate protein-coding genes models were predicted with a gene coding density of 85.9%. The SSU (16S) rRNA gene sequence of the isolate has 99% identity to Pelosinus fermentans strain DSM 17108 (Genbank accession number JF749997) and Sporotalea propionica strains TM1 and DSM13327 (FN689723 and JF749993, respectively). Since Pelosinus fermentans strains with 99-100% 16S rRNA gene similarity have been shown to have differing heavy metal-reducing characteristics, the genome sequence of this organism will provide insight into its metabolic strategies. Through the utilization of BLAST (NCBI) and KEGG a clearer understanding of carbon flow in this species has been predicted. Complete glycolysis/gluconeogenesis, pentose phosphate, mixed acid fermentation, and partial citric acid cycle pathways have been elucidated. The capacity for heavy metal tolerance, hydrogen production, and methane utilization are examples of predicted phenotypes that could impact community structure, composition, and function.

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460A Proteomic, physiological, and in silico testing of economic tradeoffs in metabolic networks Ross Carlson*, Reed Taffs, James Folsom Montana State University, United States

Microbes conserve energy and perform carbon conversions using robust and often redundant networks of enzymatic machinery. Competition for resources in nature has likely selected for phenotypes wherein operational metabolic pathways optimize tradeoffs between catabolic efficiency of an enzyme set and the investment of anabolic nutrients required to synthesize the enzymes. An in silico economics-based analysis quantified molecular-level, resource allocation tradeoff strategies that permit competitive cellular functioning under a continuum of nutrient scarcities. The analysis decomposed a metabolic network into a complete listing of non-divisible, mathematically-defined biochemical pathways which were then used to identify all strategies for investing limiting resources like iron and nitrogen into the genome encoded metabolic machinery. Nitrogen, for example, is required for the synthesis of every enzyme; as nitrogen availability decreases, tradeoff analysis predicts a testable shift from catabolism optimized phenotypes to nitrogen investment-optimized phenotypes. In silico predictions were evaluated experimentally using physiological and proteomic data collected from iron- or nitrogen-limited Escherichia coli chemostat cultures. Experimental chemostat data trends were consistent with in silico theory and illustrated that under iron- and nitrogen-limited conditions E. coli regulates its metabolism to invest scarce resource competitively at the cost of optimal biomass yields on electron donor. The study highlights a fundamental evolutionary and metabolic design paradigm for competitive network structure and control with applications to environmental microbiology, bioprocess engineering, and treatment of microbial pathogens.

461A Proteome analyses of hydrogen producing hyperthermophilic archaeon Thermococcus onnurineus NA1 in different one-carbon substrate culture conditions Young-Ho Chung1, Yoon-Jung Moon*1, Joseph Kwon1, Sung-Ho Yun1, Hye Li Lim1, Min-Sik Kim2, Sung Gyun Kang2, Jung-Hyun Lee2, Jong -Soon Choi1, Seung Il Kim1 1Korea Basic Science Institute, South Korea, 2Korea Ocean Research and Development Institute, South Korea

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is capable of H2-producing growth, considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate can be used as its carbon source remains unknown. To obtain a global view of the metabolic characteristics of H2-producing growth, the quantitative proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by combining 1D SDS-PAGE with nano UPLC-MSE method. A total of 587 proteins corresponding to 29.7% of the encoding genes were identified, and the major metabolic pathways (esp. energy metabolism) were characterized. Enzymes involved in gluconeogenesis and penotose phosphate pathways were strongly up-regulated in formate-grown cells, suggesting that formate could be utilized as a carbon source by T. onnurineus NA1. In addition, our data suggest that CO2 produced from catabolism or CO oxidation may be assimilated into the organic carbon. Overall, proteomic comparison of formate- and CO-grown cells with starch-grown cells revealed that one carbon compound, such as formate and CO, can be utilized as an efficient substrate to provide cellular carbon and/or energy by T. onnurineus NA1.

462A Genes to phenotype: establishing a physiological and metabolic basis of mixotrophy in model marine bacteria Michael Cunliffe* Marine Biological Association of the United Kingdom, United Kingdom

The Marine Roseobacter Clade (MRC) are a biogeochemically significant component of the bacterioplankton. Annotation of multiple MRC genomes has revealed that many contain carbon monoxide oxidation (cox) and inorganic sulfur oxidation (sox) genes, implying a role for the MRC in marine CO and inorganic sulfur cycling. Almost all MRC bacteria are heterotrophs, relying exclusively on organic carbon for growth, therefore CO and inorganic sulfur could be used mixotrophically as supplementary energy sources. Even though mixotrophy appears widespread in the oceans it is poorly understood at a physiological level. This research aims to better understand mixotrophy in model MRC bacteria.

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29 MRC genomes were surveyed in detail for cox, sox and associated ancillary genes. A subset of MRC bacteria were tested in the laboratory for CO- and thiosulfate-utilising capability. The physiological responses (growth rate, yield, respiration, enzyme activity, etcetera) to CO and thiosulfate as potential energy sources were studied further in the model MRC bacterium, Ruegeria pomeroyi DSS-3. Nuclear magnetic resonance (NMR)-based metabolomics was also used to establish the effects of CO and thiosulfate on metabolism in R. pomeroyi DSS-3.

The cox genes in MRC genomes fall into two distinct forms based on sequence analysis of the coxL gene; form I and form II. The two forms are unevenly distributed across the MRC genomes. Most (18/29) of the MRC genomes contain only the putative form II coxL gene. Only 10 of the 29 MRC genomes analysed have both the putative form II and the definitive form I coxL. None have only the form I coxL. Genes previously shown to be required for posttranslational maturation of the form I enzyme are absent from the MRC genomes containing only form II. Physiological analyses of a subset of nine strains revealed that only strains with both coxL forms are able to oxidise CO. All of the MRC genomes surveyed contained all the sox genes necessary for the complete transformation of thiosulfate to sulfate via the Kelly-Friedrich pathway. Apart from Dinoroseobacter shibae DFL12, all strains responded either positively or negatively to thiosulfate depending on whether they were grown on agar plates (biofilm) or in liquid media (planktonically).

Heterotrophic growth of R. pomeroyi DSS-3 was stimulated by thiosulfate, causing an increase in growth rate, yield and respiration. Conversely, even though R. pomeroyi DSS-3 rapidly oxidised CO and expressed carbon monoxide dehydrogenase, CO had not detectable physiological effect. Supporting these data, NMR-based metabolomics revealed that R. pomeroyi DSS-3 growing mixotrophically with thiosulfate is metabolically distinct from heterotrophically grown cells (glucose only), whilst, even though R. pomeroyi DSS-3 oxidised CO, there was no measurable effect on metabolism.

The physiological benefits of inorganic sulfur-based mixotrophy are apparent, yet the potential rewards of CO utilisation are yet to be elucidated. The physiology of mixotrophy in marine bacteria is clearly complex, and metabolic capability cannot be established solely on a ‘gene only’ basis. These results highlight the importance and value of experimental research with model microorganisms to validate the pathways and physiological mechanisms inferred from genomic and metagenomic data.

463A Bacterial amyloids are abundant and have multiple functions in biofilms Morten Simonsen Dueholm*1, Poul Larsen2, Mads Toft Søndergård2, Daniel Otzen3, Per Halkjær Nielsen2 1Aalborg University, Denmark, 2Aalborg University, Section of Biotechnology, Denmark, 3Aarhus University, iNANO, Denmark

Functional bacterial amyloids (FuBA) represent an interesting class of proteinaceous extracellular polymeric substances, which are encountered frequently in microbial communities from natural and engineered systems. FuBA are defined as bacterial proteins with an amyloid structure, a protein structure normally associated with protein misfolding diseases such as Alzheimer's and Parkinson's disease in humans and formation of prions in other mammals. The amyloid fold is characterized as a fibrillar tertiary structure in which the protein monomers fold as β-sheets stacked perpendicular to the fibril axis. FuBA expression is not restricted to a single bacterial phylum, but is phylogenetically broadly distributed including Proteobacteria, Bacteriodetes, Chloroflexi, Actinobacteria and Firmicutes. FuBA are not associated with a single well-defined function, but seem to include fimbriae and other cell appendages for adhesion and biofilm formation, cell envelope components, spore coating, reservoirs of cytotoxins and probably several others, yet unknown. Due to difficult handling, very few FuBA have been purified and investigated in depth. Details about the biophysical properties and ecological significance are restricted to E. coli, some pseudomonads and a few other bacteria. Here we present tools to visualize FuBA in situ and present results using these methods. A huge diversity in the location and organization of FuBA is shown within microbial communities. Methods for isolation and characterization of FuBA are also presented. The characterization includes molecular microbiology, protein biophysic and bioinformatic methods.

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464A The dark way of making oxygen Katharina Ettwig*, Daan R Speth, Joachim Reimann, Ming Liang Wu, Mike S.M. Jetten, Jan T.M. Keltjens Radboud University, Dept. of Microbiology, Netherlands

Nitric oxide (NO) and nitrous oxide (N2O) are among Nature's most powerful electron acceptors. In recent years it has become clear that microorganisms can take advantage of the oxidizing power of these compounds to convert recalcitrant aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the NC10 phylum bacterium 'Candidatus Methylomirabilis oxyfera' and the γ-proteobacterial strain HdN1 it has been suggested that under anoxic conditions with nitrate and/or nitrite, mono-oxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide only. Similarly, "aerobic" pathways for carbon degradations are employed by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite (ClO2

-). In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen formation, nitrous oxide reductase, substrate activation in the presence of nitrite was directly associated with both oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an oxygen species derived from it, in the activation reaction of methane. Although oxygen generation elegantly explains the utilization of 'aerobic' pathways under anoxic conditions, the underlying mechanism is still elusive. Here we review the current knowledge about intra-aerobic pathways, their potential presence in other organisms and propose a candidate enzymes related to quinol-dependent NO reductases (qNors) that might be involved in oxygen production.

465A The overlapping gene pair htgA/yaaW in Escherichia coli O157:H7 EDL933 Lea Fellner*1, Niklas Bechtel2, Richard Landstorfer2, Svenja Simon3, Daniela Oelke3, Siegfried Scherer2, Klaus Neuhaus2 1Technische Universitaet Muenchen, Germany, 2Lehrstuhl für mikrobielle Oekologie, Wissenschaftszentrum Weihenstephan, Technische Universitaet Muenchen, Germany, 3Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und Informationswissenschaft, Universitaet Konstanz, Germany

Overlapping genes are defined as a pair of genes whose coding regions partially or completely overlap. Overlapping genes were first observed in bacteriophages, but in bacteria they are assumed to be rare. In this work we examined the controversial overlapping gene pair htgA/yaaW in Escherichia coli O157:H7 EDL933. htgA had been described as heat inducible. Since then, the experimental evidence is scarce and htgA is now classified as (obsolete) synonym to yaaW in databases. This is astonishing insofar as htgA and yaaW are at the same locus, but in opposite orientation. Several records of microarray experiments in the databases suggest differential, strand specific regulation of the htgA/yaaW-region.

First, we analyzed the transcription of htgA and yaaW at twelve different growth conditions. Our results suggest that both, htgA and yaaW, are weakly transcribed. The latter varies with different growth conditions. Next, we cloned the suspected promoter regions of both genes upstream of a gfp reporter. We found low promoter activity upstream of htgA. yaaW seems to be organized in an operon and driven by the yaaI promoter. Third, each of the overlapping open reading frames was destroyed by introducing a mutation causing a stop codon, which is silent on the other frame. Both mutants, ΔhtgA and ΔyaaW, were tested under several growth conditions for a phenotype. The influence of different carbon sources or different inhibitory substances on the growth of the mutants was tested. Previously, a double htgA and yaaW deletion mutant has been reported to show an increased biofilm formation. Thus, we investigated the biofilm formation of the mutants as well. Interestingly, both mutants showed a slightly increase in biofilm formation. Fourth, we expressed yaaW, but were not successful to express htgA using an expression vector.

The promoter activities upstream of htgA and yaaW and the increased biofilm formation of both mutants give hints that htgA and yaaW could indeed be an overlapping gene pair. Further investigations are necessary to ascertain the concrete functions of these two putative genes. Since we could not express HtgA, this protein is either present in a very low amounts or is unstable.

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466A Ecology and evolution of a hybrid isoprenoid-producing actinomycete lineage Kelley Gallagher*, Paul Jensen Scripps Institution of Oceanography, United States

Secondary metabolites are sometimes created by the fusion of products derived from different biosynthetic pathways. One example is a class of compounds called hybrid isoprenoids, which generally consist of a peptide, polyketide, or phenazine skeleton to which an isoprene moiety is appended. At least thirty-six distinct hybrid isoprenoids have been identified from bacteria. While many of these have significant bioactivity, none have a known natural function for the producing organism. Actinomycetes are a group of high G+C bacteria that are well known for the production of secondary metabolites. Hybrid isoprenoid production is rare among the actinomycetes with the exception of a robust lineage of marine sediment-derived strains called MAR4. Collectively, eight diverse hybrid isoprenoid classes have been observed from MAR4 strains, with some strains producing two or three different classes. This ability is unmatched in the prokaryotic world, making this an ideal group for studying the ecology and evolution of hybrid isoprenoids. To better assess the diversity of MAR4 strains, both culture and culture-independent approaches were used on sediments collected off the Channel Islands, California. Cultivation efforts led to the isolation of five new MAR4 phylotypes. These strains are now being studied as a source of new hybrid isoprenoids. Diversity observed exclusively in the clone libraries suggests that continued cultivation efforts are warranted. While the ecological functions of hybrid isoprenoids remain unknown, they often resemble the redox-active diffusible electron shuttles produced by Gram-negative bacteria. In addition, a MAR4 strain grown in a microbial fuel cell produces a sustained current in aerobic conditions, evidence that these strains are capable of extracellular electron transfer. A series of bioreactor experiments were performed to test the hypothesis that MAR4 hybrid isoprenoid production increases under low oxygen conditions. The results show that compounds with a phenazine skeleton are upregulated in microaerophilic conditions, and these molecules are now being studied as candidate electron shuttles.

467A Reduction of methacrylate by Geobacter sulfurreducens AM-1 - new property relevant for industrial wastewater treatment Alexander Galushko*1, Anna Obraztsova2, Kestas Laurinavichius3, Oxana Arkhipova3, Galina Mikoulinskaia4, Fred Rainey5, Vasilii Akimenko6 1Vienna University, Austria, 2Synthetic Genomics, USA, 3Institute Biochemistry Physiology Microorganisms RAS, Russia, 4Institute Bioorganic Chemistry RAS, Russia, 5University of Alaska, USA, 6Institute Biochemistry Physiology Microorganisms RAS, USA

Methyl ester of methacrylate (MEMA) has many industrial applications including the production of poly(methyl methacrylate) plastics (for ex. plexiglas). Wastewater of MEMA-based industry is often contaminated by this compound. We investigated the mechanism and pathway of microbial degradation of MEMA under methanogenic conditions. MEMA was converted to methane via several successive steps. The key step in the conversion of MEMA was reduction of methacrylate to isobutyrate after MEMA hydrolysis. We got interested in the microorganism that performed the reduction of methacrylate to isobutyrate. A pure culture of rod-shaped bacterium, strain AM-1, was isolated that grew by coupling acetate oxidation to the reduction of methacrylate. Acetate was oxidized to carbon dioxide via reactions of the citric acid cycle. The putative methacrylate reductase of strain AM-1 was shown to be a periplasmic flavin-containing protein that did not reduce fumarate. Growth yield of strain AM-1 on acetate with methacrylate was considerably lower than on acetate and fumarate. The type strain PCA of Geobacter sulfurreducens was the closest cultivated relative based on 16S rRNA gene similarity. However, strain PCA was not able to reduce methacrylate to isobutyrate. A gene that coded a protein with the N-terminal amino acid sequence similar to the putative methacrylate reductase of strain AM-1 was not found in the genome of strain PCA. Geobacter species form a group of bacteria with broad environmentally relevant properties of the metabolism. So far, the property to grow using methacrylate as the terminal electron acceptor seemed to be a new for Geobacter cluster and unique for strain AM-1.

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468A The role of C4 metabolism in the marine diatom Phaeodactylum tricornutum Maya Haimovich-Dayan*1, Nitsan Garfinkel1, Daniela Ewe2, Yehouda Marcus3, Ansgar Gruber2, Peter G. Kroth2, Aaron Kaplan1 1Hebrew University, Israel, 2Universität Konstanz, Germany, 3Tel Aviv University, Israel

Diatoms are important players in the global carbon cycle. The apparent photosynthetic affinity for ambient CO2 is 100-fold higher than that of the ribulose 1,5-bisphosphate carboxylase/oxygenase indicating that a CO2-concentrating mechanism is functioning in these organisms. 14C labeling experiments and presence of complete sets of genes essential for the biochemical C4 in two diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, supported the notion that CO2 fixation occurs via this pathway. However, the nature of the CCM - either a biophysical or a biochemical C4-pathway - remains elusive. Our purpose is to understand the nature of the CCM functioning in these organisms. We used RNA-interference to silence the single gene encoding pyruvate-orthophosphate dikinase in P. tricornutum, an essential enzyme in the C4 metabolism that converts pyruvate into phosphoenolpyruvate. The mutants obtained possess much lower ppdk transcript and PPDK activity but the photosynthetic rate under limiting inorganic carbon level was hardly affected. Further, even in the wild the PPDK activity is far too low to support the observed photosynthetic rate. These data clearly indicate that the C4 route is not essential for the CCM activity. The photosynthetic Vmax was slightly reduced in the mutant possibly reflecting a metabolic constrains that resulted in a larger lipid accumulation in the mutants. We propose that C4 metabolism does not serve the purpose of CO2 concentration in P. tricornutum but instead is important for photoprotecion and pH homeostasis. Pulse amplitude modulated measurements revealed a faster non-photochemical fluorescence quenching in the mutants possibly compensating for the loss of ability to dissipate excess energy through a futile C4 route.

469A Polyphosphate storage in response to nitrogen limitation in Beggiatoa alba Sandra Havemeyer*, Heide Schulz-Vogt Max Planck Institute for Marine Microbiology, Germany

Polyphosphate storing microorganisms are of special interest, because they can have a direct influence on phosphorus cycling, e.g. by releasing high amounts of phosphate from intracellular stored polyphosphate. This reaction can result in an oversaturation of phosphate in sediment pore water, which leads to the spontaneous precipitation of phosphorus-rich minerals. For such a process to occur it is not sufficient that a polyphosphate accumulating bacterium is present, but the environmental conditions have to favour polyphosphate storage and subsequently polyphosphate degradation. In a recent study, it was shown, that a marine lithotrophic Beggiatoa strain can release high amounts of phosphate from intracellular stored polyphosphate in response to anoxia and high sulfide concentrations (Brock and Schulz-Vogt, 2011). In this study we investigated the polyphosphate storage of the closely related heterotrophic freshwater Beggiatoa alba strain B15LD in order to compare the physiology of polyphosphate of these two related organisms inhabiting very different environments.

We cultivated Beggiatoa alba in two different heterotrophic liquid media and we observed that in medium A (according to Strohl and Larkin, 1978) they do store polyphosphate while they do not when grown in medium B (according to Schmidt et al., 1987). In order to find out which difference between the media is responsible for polyphosphate storage, the concentrations of the components in medium B were adapted step by step to ones in medium A and the filaments were examined for polyphosphate after growth in the modified medium B. Polyphosphate was visualized and quantified by staining with 4',6-Diamidin-2'-phenylindole (DAPI), a common stain for DNA. It shows a shift from the blue DNA signal with an emission wavelength around 460 nm to a yellow emission around 525 nm when binding to polyphosphate.

We observed that high ammonium or high nitrate concentrations inhibited polyphosphate storage. In contrast, cultures which were nitrogen limited stored high amounts of polyphosphate. From this we conclude that freshwater Beggiatoa may not have an effect on phosphorus sequestration in environments with high nitrogen concentration due to almost no polyphosphate storage. Moreover, despite their close phylogenetic relationship, the freshwater Beggiatoa alba strain stores polyphosphate at different conditions compared to the marine Beggiatoa strain, which does not store less polyphosphate in response to increasing nitrogen concentrations in the medium. Apparently, even

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closely related bacteria may use polyphosphate for very different purposes, depending on the habitat and the physiology. Furthermore, our results demonstrate once more that polyphosphate storage is not always a feature which is present or absent in a certain microorganisms, but can largely depend on different factors introduced by the composition of the medium or the environment.

470A Characterization of novel sulfate-reducing bacteria isolated from tidal flat sediment Yuriko Higashioka*1, Hisaya Kojima2, Miho Watanabe2, Manabu Fukui2 1Kochi National College of Technology, Japan, 2Institute of Low Temperature Science, Hokkaido University, Japan

Sulfate-reducing bacteria are anaerobes which utilize organic compounds and hydrogen as electron donors. They usually prefer relatively low-molecular-weight organic substrate such as short chain fatty acids. Some strains of sulfate-reducing bacteria can also degrade hydrocarbons. It has been reported that some alkane, alkene, or mono-aromatic hydrocarbons can be degraded by sulfate-reducing bacteria. However, there are not so many pure cultures of them. Therefore, it is expected that to obtain more pure cultures provides the information regarding hydrocarbon biodegradation under obligatory anaerobic condition as well as diversity of sulfate-reducing bacteria. Recently, a novel strain of toluene-degrading sulfate reducer was isolated from an enrichment culture established with tidal flat sediment as inoculum. In the process of isolation of that strain, another bacterium with characteristic 16S rRNA gene sequences was also enriched. In the present study, the bacterium was isolated in a pure culture as strain S28bF, designated as type strain of a novel species in a new genus, Desulfatitalea tepidiphila. Two other strains of the same species, S28OL1 and S28OL2, were also isolated from the same sediment. Strain S28bF was isolated via series of cultures with several times changes of substrate for growth, crude oil, p-xylene, benzoate, and toluene. In the final step, strain S28bF was separated by repeated serial dilution using fumarate as substrate. The others, S28OL1 and S28OL2, were isolated from crude oil-degrading enrichment culture by agar shake dilution using sodium lactate as the substrate. The temperature ranges for growth of the three strains were 25-43ºC (S28bF), 18-45ºC (S28OL1), and 13-42ºC (S28OL2), respectively. These temperatures are higher than those in the sampling site of the inoculum ranging from 6.5 to 32.8ºC, and the incubation temperature during enrichment and isolation, 28ºC. The isolates used sulfate and thiosulfate as electron acceptors, but not nitrate. Three novel strains are closely related to each other with 16S rRNA gene sequence similarities of 99%, and belong to the family Desulfobacteraceae within the class Deltaproteobacteria. The closest relative was Desulfosarcina cetonica (similarity equals 93.3%). The analysis targeting functional genes which involved in sulfate respiration, dsrA and aprA, also showed three strains belong to the family Desulfobacteraceae and phylogenetically distinct from other members of Desulfobacteraceae including the genus Desulfosarcina. Desulfosarcina species are known to oxidize aromatic compounds such as benzoate, but novel strains could not grow on benzoate. Additionally the fatty acid profile of strain S28bF had some distinct features in comparison to Desulfosarcina variabilis, that is, more various and abundant anteiso-fatty acids, fewer variations and lower content of unsaturated fatty acids, and occurrence of the longer chains with 19-20 carbons. Although the isolates were obtained from enrichment cultures supplemented with hydrocarbons, they could not utilize toluene and n-hexane. In addition, benzene, o-xylene, m-xylene, p-xylene, ethylbenzene, and n-decane could not support growth of strains 28bF. Three novel isolates of the Desulfatitalea tepidiphila were obtained from tidal flat sediment, and they are characterized by preference for moderately warmth, distinct features of fatty acid profiles, and no degradability of hydrocarbons.

471A Salmonella Stn regulates membrane composition and integrity Toshiya Hirayama*, Masayuki Nakano Nagasaki University, Japan

It has been shown that Salmonella enterotoxin (Stn) is considered a Salmonella virulence factor and a responsible for the enterotoxicity of Salmonella. However, the role of Stn in Salmonella virulence is still debated because it had been reported that the contribution of Stn in the virulence is variable in each strain. In this study, to evaluate the functions of Stn, we examined the relationship between Stn and Salmonella virulence using the stn gene-deleted mutant in in vitro and in vivo models.

S. Enteritidis strain 171, a clinical isolate from Thailand, was used as a standard strain. The mutant strain was constructed by homologues recombination. To evaluate the functions of Stn, we examined

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the virulence phenotypes of mutant strain using cultured mammalian cells and by a murine ligated ileal loop model. In addition, we also performed the protein profiles of mutant strain.

We found that the stn gene was specifically distributed in Salmonella spp. irrespective of their serotypes and other enteropathgens did not possess this gene. Although we examined Salmonella virulence in in vitro and in vivo models, we did not observed remarkable difference of virulence phenotypes between wild-type and mutant strain. We next characterized the phenotypes of mutant strain in various in vitro conditions. When we analyzed the proteomic profile of total Salmonella cell membrane, it was remarkable for the absence of a protein signal in the mutant strain, which was identified as OmpA. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy and OmpA was localized by immuno-gold labeling.In this system, Stn, affected membrane morphology. These results indicate that Stn, via regulation of OmpA localization, functions in the maintenance of membrane integrity.

472A The poly(ethylene succinate) (PES)degradation of thermophilic actinomycetes 06174T-1 in soil Kim Chi Hoang*1, Min Tseng2 1Ta Hwa Institute of Technology, Taiwan, 2Bioresource Collection and Research Center, Food Industry Research and Development Institute, Taiwan

In this study, we investigated the degradation of PES at 50℃ by a thermophilic actinomycete isolate, a Laceyella sacchari sp. strain 06174T-1. The strain 06174T-1 was able to degrade PES films within 10 days in liquid cultures and in soil at 50℃. Furthermore, the films of PCL and PLA were degrade at 50℃ in liquid cultures and in soil in this study.

Thermophilic actinomycetes strains were isolated from various environments in Taiwan and the strain 06174T-1 screened for degradation of polyethylene succinate (PES) by the clear-zone method. The strain 06174T-1 was able to degrade PES films within 10 days in liquid cultures and in soil at 50℃. On the basis of morphological and chemical characteristics, the strain 06174T-1 belong to the genus Laceyella sacchari.

The addition of the thermophilic strain in soil is an efficient process for biodegradation of bio-based plastic.

473A Bacterial degradation of steroid compounds - new insights into the side chain degradation pathway Johannes Holert*1, Onur Yücel2, Žarko Kulić2, Bodo Philipp1 1University of Muenster, Germany, 2University of Konstanz, Germany

Steroids are ubiquitous natural compounds with diverse functions in eukaryotic organisms. They enter the environment mainly via excretion by and decay of animals and plants. In bacteria, steroids occur only as rare exceptions but the ability of transforming and degrading steroids is widespread among different phylogenetic groups of bacteria.

We investigate bacterial steroid degradation using the bile salt cholate as a model compound and Pseudomonas sp. strain Chol1 as a model organism. Cholate degradation is initiated by oxidative reactions at the A-ring followed by cleavage of the acyl side chain attached to C17. Mutants of strain Chol1 with defects in the genes skt and acad are defect in degradation of the acyl side chain. In culture supernatants of the skt mutant, (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) and 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO) and in culture supernatants of the acad mutant 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) accumulate as dead end products. The structure of these compounds strongly suggests that degradation of the acyl side chain proceeds via β-oxidation. However, explicit in vitro data was missing so far.

Therefore, we investigated the degradation of the acyl side in vitro CoA-activation assays using purified cell extracts of strain Chol1 in the presence of CoA and ATP as well as the electron acceptors NAD+ and phenazine methosulfate.

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When cholate or Δ1,4-3-ketocholate were used as substrates, Δ1,4-3-ketocholyl-CoA accumulated as end product indicating that further oxidation of the acyl side chain was not possible in vitro under the applied conditions.

When DHOCTO was used as substrate, the complete side chain was cleaved off in vitro in the presence of NAD+ leading to 7α,12α-dihydroxy-androsta-1,4-diene-3,17-dione (12α-DHADD) and 7α-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products. The CoA-ester of DHOPDC accumulated transiently in the assay. The same products were formed, when DHOPDC was used as substrate.

Without adding NAD+, a new product was formed exclusively from DHOCTO but not from DHOPDC, which was identified by MS and NMR analysis as 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA). This product had a shortened C3-side chain with an aldehyde function at C22. This intermediate was completely transformed to DHOPDC and DHOPDC-CoA upon subsequent addition of NAD+ to the assay. When purified DHOPDCA was used as substrate in in vitro assays containing only purified cell extract of strain Chol1, it was completely transformed into DHOPDC upon addition of NAD+.

In conclusion, DHOPDCA is a new and unexpected intermediate of the side chain degradation pathway of cholate by Pseudomonas sp. strain Chol1, in which DHOCTO is activated by CoA followed by a hydratase reaction forming THOCDO. In a putative aldolase reaction acetyl-CoA is split off forming DHOPDCA which is further oxidized to DHOPDC by a putative NAD+-dependent aldehyde dehydrogenase. We conclude that shortening of the C5 side chain to a C3 side chain by Pseudomonas sp. strain Chol1 does not occur by a typical β-oxidation cycle, but rather by a CoA-dependent hydratase/aldolase pathway.

474A Expression of genes involved in aerobic degradation of dichloromethane by Xanthobacter autotrophicus under different nutritional conditions Akifumi Hosoda*, Hiroto Tamura Meijo University, Japan

Halogenated aliphatic compounds including dichloromethane (DCM) are frequently occurring pollutants by using many industries. Microbial degradation of these compounds is important for the treatment of pollutants. Xanthobacter autotrphicus GJ10 (ATCC43050) is well known as a degrader of haloalkanes including 1,2-dichloroethane (DCE). The haloalkane dehalogenase (DhlA), which response with high affinity to DCE but low affinity to DCM, has been isolated from strain GJ10. In this study, we investigate the effect of nutrient additives for DCM degradation to apply practical use. Furthermore, the expression of genes involved in DCM degradation was observed to access the DCM degradation activity.

Aerobic degradation of DCM was performed with minimum salt medium (MS) containing 2.5 mM DCM. Strain GJ10 was inoculated into test tubes containing 5 mL of MS, and incubated at 27°C for 21 days. All test tubes were sealed with sterilized Teflon coated butyl rubber stoppers. To enhance the degradation activity, some nutrients {0.1% (v/v) of biostimulation agent, BD-C and 1 mM acetate} were added to the MS. A 5 mL aliquot was collected from treated samples after a specified period and immediately extracted with an equal volume of hexane to determine DCM content by capillary gas chromatography equipped with electron capture detector. Volatile fatty acids were quantified by high-pressure liquid chromatography equipped with an electrical conductivity detector. The bacterial growth was monitored by measurement of the optical density at 660 nm. Total RNAs were extracted from tested cells with PicopureTM RNA isolation kit. cDNAs were synthesized by High Capacity cDNA Reverse Transcription kit from 0.1 mg of total RNAs. To investigate the gene expression involved in DCM degradation, Real-Time PCR was carried out to quantify the some metabolic genes including dhlA and 16S rRNA gene. The fold change in the target gene normalized to 16S rRNA genes was calculated for each sample using ΔΔCT method.

Strain GJ10 was able to degrade 1.5 mM of DCM in MS supplemented with BD-C for 21 days incubation. DCM degradation rate was calculated to be 0.068 mM/day. On the other hand, the degradation of DCM was reached to 2.2 mM (0.10 mM/day) in MS treated with BD-C and acetate. Formate, which was metabolite during DCM degradation, was increased to 2-fold higher (5 mM) than

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theoretical production during incubation. Acetate was immediately assimilated for 3 days incubation, but accumulated after 10 days incubation. These changes suggest that formate and acetate were derived from BD-C during DCM degradation. The dhlA gene level was10-fold increase in BD-C and acetate treatment during degradation of DCM, whereas those in only BD-C treatment was decrease.

In conclusion, aerobic DCM degradation by X. autotrphicus strain GJ10 is enhanced by acetate addition. Moreover, acetate would be contributed to DhlA expression. Further analysis of other metabolic genes expression will be needed to elucidate the role of acetate in DCM metabolism of strain GJ10.

475A Characterization of tert-butyl-alcohol-hydroxylase, a Rieske oxygenase in MTBE degradation pathway of Methylibium petroleiphilum PM1 Krassimira Hristova*1, Anthony Kappell1, Jian Sun2 1Marquette University, United States, 2China Agricultural University, China

In Methylibium petroleiphilum PM1, two different oxygenase enzymes are involved in the aerobic degradation of the groundwater pollutant methyl tert-butyl ether and its downstream metabolite, tert-butyl alcohol (TBA), which is a potential carcinogen. PM1 is an environmentally important methylotrophic bacterium capable of completely oxidizing MTBE and TBA to CO2. Genetic evidence that a flavin-dependent monooxygenase, MdpA, is directly responsible for MTBE hydroxylation to TBA was previously reported. Gene disruption studies have established that conversion of TBA to hydroxy-methyl propanol is catalyzed by the two-component enzyme system consisting of the Rieske non-heme oxygenase, encoded by mdpJ, and a NAD(P)H dependent Fe-S reductase, encoded by mdpK. The aim of this study was to overexpress and purify the two components of TBA monooxygenase to biochemically characterize their function.

The genes encoding MdpJ and MdpK were cloned into the pETDUET vector or a pET28a derivative for overexpression and purification in E. coli. The recombinant proteins have been purified under reducing conditions (1 mM dithiothreitol) with binding to the Ni2+ column and subsequent purification performed under anaerobic conditions at 4°C. Observations of enzyme activity was performed by measuring oxygen consumption using a Clark-type electrode.

Overexpression and purification of the MdpJ, the oxygenase, and MdpK, the reductase, was successful based on bands observed on SDS-PAGE corresponding to the molecular size of the proteins, 53 kDa and 37 kDa respectively, in conjunction with purification tags. The UV-Vis absorption spectrum of the oxidized and reduced MdpJ protein confirmed the presence of a Rieske-type (2Fe-2S) center in the MdpJ. FPLC analysis of the purified MdpJ protein suggest the formation of a trimeric complex. Preliminary measurements of oxygenase activity from MdpJ in air-saturated buffer containing an artificial electron donor system indicate that MdpJ was able to utilize TBA at the micromolar range. Under the same conditions reaction rates for MTBE were negligible at milimolar range.

In laboratory microcosms and field studies inoculation with M. petroleiphilum PM1 culture substantially reduced TBA formation. Detailed knowledge about the biochemical properties of TBA monooxygenase thus is highly relevant in the fields of biodegradation and bioremediation of persistent groundwater pollutants.

476A Metabolic adaptations in microbial communities in the Baltic Sea region Karolina Ininbergs*1, John Larsson1, Christoper L Dupont2, Shibu Yooseph2, Johannes Goll2, Mathangi Thiagarajan2, Narin Celepli1, Johannes Asplund-Samulesson1, Björn Brindefalk1, Birgitta Bergman1 1Stockholm University, Sweden, 2J. Craig Venter Institute, USA

The Baltic Sea is a semi-enclosed brackish water sea with steep horizontal gradients in environmental parameters such as salinity and nutrients, with more limnic conditions in the north and increasing salinity towards the south. Metagenomic analysis of microbial communities from 21 samples collected at 11 different locations along this salinity gradient (including arctic lake Torne Träsk and the Swedish west coast) revealed significant differences in number of reads related to different metabolic pathways (Torne Träsk, Bothnian Sea, Bothnian Bay, Baltic Proper and West Coast). Out of 151 KEGG pathways 27 varied significantly between basins. We observed a significant decrease of reads related

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to oxidative phosphorylation (OP), the major source of ATP in non-photosynthetic cells, from Torne Träsk, through the Bothnian Bay to the marine Baltic West stations. The highest proportions of OP reads were found in the largest microbial fractions (3.0-200 µm) from surface waters of lake Torne Träsk, while there was no significant difference between size fractions (down to 0.1) in general. Although several of the 12 enzymes involved in the OP pathway displayed a decreasing trend from the basins with lower salinity towards the higher salinity basins the only enzyme which differed significantly in abundance was polyphosphate kinase (ppk). There was no obvious trend in relative abundance of the ppk enzyme related to either N:P ratio or PO4 concentrations. However, several of the sampling sites with the highest relative abundance of ppk show constant low PO4 concentrations on an annual basis. The opposite trend was found for sampling sites with large seasonal fluctuations in PO4 concentrations, with significantly higher concentrations during spring and fall compared to summer. The difference in ppk abundance may be a result of chronic phosphate limitation experienced by microbes in the northern areas of the Baltic Sea, leading to a higher need to build up polyphosphate concentrations to endure long periods of P-starvation.

477A Comparative genomic analysis and BTEX degradation pathways of Pseudoxanthomonas spadix BD-a59 Che Ok Jeon, Eun Jin Choi*, Hyun Mi Jin, Renukaradhya K. Math Chung-Ang University, South Korea

Pseudoxanthomonas spadix BD-a59, isolated from a gasoline-contaminated site, is able to degrade all six BTEX compounds (benzene, toluene, ethylbenzene, o-, m-, and p-xylene) that are known as common groundwater and soil contaminants. The genomic features of strain BD-a59 were analyzed bioinformatically and compared with those of Pseudoxanthomonas suwonensis 11-1. The genome of strain BD-a59 was differentiated from that of strain 11-1 in many genetic features; GC skew, GC contents, karlin signature skew, numbers of phage integrase, dioxygenase, and monooxygenase, percentage of the cluster of orthologous groups, and numbers and characteristics of genomic islands (GIs), etc. High abundances of phage integrases and GIs indicated that the genome of strain BD-a59 might have high chances of horizontal gene transfers (HGT), phage attacks, and genetic reshufflings in its evolutionary history. It was revealed that the degradation genes of benzene/toluene, ethylbenzene, and xylene were coded on GIs 9, 13, and 21, respectively, suggesting that they might be recruited by HGT. We estimated biodegradation pathways of six BTEX compounds bioinformatically, which were confirmed by the analysis of intermediates of each BTEX compound using GC-MS. In conclusion, high abundances of dioxygenases, monooxygenases, and hydroxylases, in strain BD-a59 likely confer ecological fitness traits of aromatic compound degradations. This work was supported by the Next-Generation BioGreen 21 Program (No. SSAC2011-PJ008220), RDA, Korea.

478A Involvement of the anaerobic respiration of Shewanella Oneidensis MR-1 in the stability of extracellular U(VI) nanowires Shenghua Jiang, Sunhwa Park*, Hor-Gil Hur Gwangju Institute of Science & Technology, South Korea

Uranium (VI) is considered to be one of the most widely dispersed and problematic environmental contaminants, due in large part to its high solubility and great mobility in natural aquatic systems. We previously reported that under anaerobic conditions, Shewanella oneidensis MR-1 grown in the medium containing uranyl acetate rapidly accumulate long, extracellular, ultrafine U(VI) nanowires composed of polycrystalline chains of discrete meta-schoepite (UO3·2H2O) nanocrystallites. The wild type MR-1 finally transformed the uranium (VI) nanowires to uranium (IV) nanoparticles via the further reduction. In order to investigate the influence of the respiratory chain in the uranium transformation process, a series of mutant strains lacking a periplasmic cytochrome MtrA, outer membrane (OM) cytochrome MtrC and OmcA, a tetraheme cytochrome CymA anchored to the cytoplasmic membrane, and a trans-OM spanning-barrel protein MtrB, were tested in this study. Although all the mutants produced U(VI) nanowires like the wild-type did, the rates of the transformation from U(VI) nanowires to U(IV) nanoparticles were various, especially the mutant with deletion in the tetraheme cytochrome CymA stably maintained the uranium(VI) nanowires, suggesting that the respiratory chain of S. Oneidensis MR-1 is probably involved in the stability of extracellular U(VI) nanowires which might be easily treated via physical processes of filtration or flocculation for the remediation of uranium contamination in sediments and aquifers, as well as the recovery of uranium in manufacturing processes.

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479A Dichloromethane degradation in sediments and identification of a dichloromethane-fermenting Dehalobacter sp. Shandra D. Justicia-Leon*1, E. Erin Mack2, Frank E. Löffler3 1Georgia Institute of Technology, United States, 2DuPont Corporate Remediation Group, United States, 3University of Tennessee, United States

Dichloromethane is produced at a scale of several million tons per year in the United States and used in a variety of industrial processes. Extensive use and improper disposal practices have caused dichloromethane to become a common groundwater contaminant. In addition to direct release, halomethanes are formed via natural processes such as brush and vegetation burning, volcanic activity, and by marine phytoplankton and some macroscopic algae. Microorganisms that exploit dichloromethane as a source of energy or carbon in aerobic processes have been characterized, but dichloromethane often resides in anoxic environments. Anaerobic catabolism of dichloromethane has been observed in cultures of Dehalobacterium formicoaceticum, a homoacetogenic bacterium. Although much research has been conducted to understand the biological mechanisms underlying the anaerobic transformation of dichloromethane, the diversity and distribution of dichloromethane-degrading microorganisms in anoxic environments is unclear, a knowledge gap that limits our ability to predict and control the fate of dichloromethane. To address these shortcomings, sediment samples from 45 geographically distinct locations were used to establish microcosms in 20-mL glass vials containing 12 mL of sediment slurry amended with 0.3 mM dichloromethane and 30 mM bicarbonate-buffered basal salts medium. Dichloromethane was completely consumed after 5 weeks in microcosms from 13 sites, and consumption rates increased upon consecutive dichloromethane amendments. The transfer of supernatant from active microcosms to fresh medium yielded one sediment-free, dichloromethane-degrading enrichment culture, in which acetate and inorganic chloride were detected as dichloromethane degradation products. Reductive dechlorination (that is the formation of chloromethane) was not observed and the culture failed to transform chloromethane. 454 pyrosequencing of the V3 through V1 regions of 16S rRNA gene amplicons obtained with template DNA of the dichloromethane-degrading culture, as well as the analysis of 16S rRNA gene clone libraries, indicated that a Dehalobacter sp. predominates in the enrichment culture. Quantitative growth measurements demonstrated that the Dehalobacter cell numbers increase concomitant with dichloromethane degradation. No Dehalobacter growth occurred in controls inoculated with the dichloromethane-degrading enrichment culture but without dichloromethane addition. A 16S rRNA gene-based quantitative polymerase chain reaction assay distinguished the dichloromethane-degrading Dehalobacter sp. from the characterized tetrachloroethene-respiring Dehalobacter restrictus, and was used to evaluate the distribution of dichloromethane-fermenting Dehalobacter sp. in river sediments. These findings imply that Dehalobacter growth is not limited to organohalide respiration and that Dehalobacter spp. contribute to the turnover of dichloromethane in sediments.

480A Optimization of culture conditions for the production of butanol from sweet sorghum juice by Clostridium beijerinckii SBP2-HB Miho Kanemoto*1, Manami Akiyama2, M. Habibur Rahman2, Yoshinori Sato3, Shigeru Chohnan2, Youji Nitta2, Yasurou Kurusu2, Hiroyuki Ohta2 1Tokyo University of Agriculture and Technology, Japan, 2Ibaraki University, Japan, 3National Research Institute for Cultural Properties, Tokyo, Japan

Sweet sorghum, Sorghum bicolor (L) Moench has been identified as a possible bio-fuel feedstock because of its fermentable sugar-rich biomass yield under a wide range of environmental conditions. Since its low nutrition and water demands, this plant is considered highly suited to design and develop the bio-fuel production system in recently increasing abandoned agricultural areas around the urban in Japan. In our previous study, bacteria able to produce butanol from sweet sorghum juice (SSJ) were isolated from a soil and a 2.4% (v/v) butnaol-tolerant strain (SBP2-HB), identified as Clostridium beijerinckii was selected. Here we report the characteristics of strain SBP2-HB and its culture conditions suitable for butanol production from SSJ. Two sweet sorghum varieties, FS902 (Big sugar sorgo, Snow Brand Seed Co., Ltd.) and KCS105 (Super sugar sorgo, Kaneko Seeds Co., Ltd.), were cultivated to prepare SSJ in our university farm, Japan. The strain was grown anaerobically in N2-gassed bottles containing diluted SSJ media supplemented with 0.03% (w/v) cysteine hydrochloride. Growth of the strain was observed at temperatures ranging from 20 to 35 ºC with an optimal temperature of 35 ºC. Because the SSJ contained high sugar concentrations, approx. 140 mM glucose, 140mM, fructose, and 200 mM sucrose, the strain was gown in 1.2- to 15-fold diluted SSJ to examine the relationship between sugar utilization and butanol production. The glucose effect, more

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exactly catabolic repression, was evident in the growth of strain SBP2-HB in the FS902-SSJ and the utilization of sucrose was not found when residual monosaccharides were detected in the culture. The total amount of monosaccharides used for the growth in the FS902-SSJ was 100mM and thus the utilization of sucrose was found in the diluted SSJ containing <100 mM of initial monosaccharides. In the case of KCS105-SSJ, the total amount of monosaccharides used for growth was the same as the FS902-SSJ (100 mM), but a higher initial concentration of monosaccharides (180mM) was reqired to support the use of 100mM monosaccharides, suggesting the lower efficiency of monosaccharides in the KCS105-SSJ. Therefore, the sucrose utilization occurred in the diluted KCS105-SSJ containing <180 mM of initial monosaccharides. When strain SBP2-HB was grown in diluted SSJ, the butanol yield (mol butanol produced/mol monosaccharide consumed) was 0.86 and 0.73 in SSJ prepared from cultivar KCS105 and FS902, respectively. Collectively, the strain showed the maximum butanol production and sugar consumption in the SSJ containing about 300 mM of initial monosaccharide-base sugar concentration. Further studies are now in progress to grow the strain in chemostat cultures to examine its metabolic characteristics.

481A Evidence of hybrid N2O production from NH2OH and NO2- in cell suspension of

oligotrophic ammonia-oxidizing bacterial strain Nitrosomonas sp. AL212 using a 15N and 18O tracer technique Chie Katsuyama*1, Keisuke Koba2, Yuichi Suwa1 1Chuo University, Japan, 2Tokyo University of Agriculture and Technology, Japan

Ammonia-oxidizing bacteria (AOB) have been shown to produce nitrous oxide (N2O) from hydroxylamine (NH2OH) during ammonia oxidization and/or from nitrite (NO2

-) during nitrifier denitrification under aerobic condition. N2O can be also formed chemically by auto-oxidation of NH2OH and by reduction of NO2

- with NH2OH, which is designated as chemodenitrification. In addition, the formation of hybrid N2O (co-denitrification) from NH2OH and NO2

- was reported mainly in denitrifying species. The aim of this study is to determine which mechanisms are responsible for producing N2O by an oligotrophic AOB species using a 15N and 18O tracer technique. Cell suspension of genome-analyzed Nitrosomonas sp. AL212, which is sensitive to ammonium at levels >30 mM and belongs to cluster 6A of AOB, was incubated with NH2OH and 15N-labeled or 15N- and 18O-labeled NO2

- under aerobic condition. Produced N2O and its fragments (m/z 44, 45, 46, 47, 48, 28, 29, 30, 31, 32, and 33) were analyzed using quadrupole GC/MS. N2O was produced during aerobic incubation with NH2OH and 15NO2

-/15N18O2- in the medium with and without cells. 14,15N2O produced in cell suspension was

significantly higher than that in cell-free medium. The ratio of 14N15NO (internal 15N) or 15N14NO (external 15N) in 14,15N2O is approximately 0.5. Cell suspension produced 14,15N2O more than 100 times higher than 15,15N2O. The 15N isotope ratio of the net N2O production from cell suspension during early incubation period was ca. 40 atom%. 15N of added NH2OH and NO2

- was 0.37 and 98 atom%, respectively. These results suggested that strain AL212 cell suspension formed hybrid N2O from NH2OH and NO2

- via co-denitrification rather than N2O solely from NO2- via nitrifier denitrification under

aerobic condition. When the AL212 cell suspension was incubated with NH2OH and dual-labeled 15N18O2

- (18O >90 atom%), instead of 15NO2-, 18O-containing N2O was produced, confirming hybrid N2O

formation with the AOB strain. The 18O isotope ratio suggested that 18O exchange between H2O and intermediates of the hybrid N2O production pathways could occur during NO2

- reduction. The 15N and 18O tracer technique would be powerful tool to elucidate N2O-producing pathway.

482A Proteomic characterization of polycyclic aromatic hydrocarbon(PAHs) - utilizing marine bacterium, Novosphingobium pentaromativorans US6-1 Seung Il Kim*1, Sungho Yun1, Young Ho Chung1, Kae Kyoung Kwon2 1Korea Basic Science Institute, South Korea, 2Korea Ocean Research & Development Institute, South Korea

Contamination of marine sediments with polycyclic aromatic hydrocarbons (PHAs) is critical environmental problem because PHAs have toxic, carcinogenic, and recalcitrant properties. Therefore, biodegradation of PAHs from contaminated environments is very important. Halophilic marine bacterium, Novosphingobium pentaromativorans US6-1 is a gram negative bacterium and has an ability to degrade several PAHs such as pyrene, benzo[a]pyrene, phenanthrene, et al. Recently, genome sequencing of US6-1 was finished and the draft data is available. Purpose of this project was to search for enzymes of PHAs biodegradation pathways in stain US6-1. In this study, proteomic research of stain US6-1 cultured under several PAHs was performed using shot-gun method such as 1DE-LC-MS/MS analysis. We confirmed major bio-degrading genes cording the aromatic-

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hydrocarbon-degrading proteins was located in the large plasmid of strain US6-1 using genomic & proteomic analysis. Interestingly, this strain uses extradiol cleavage pathway (catechol-2,3 dioxygenase pathway) for the degradation of mono-aromatic compounds. Identification of these proteins and their biological significance were presented. We report the PHAs biodegradation pathway and physiological characterization of stain US6-1 in this study.

483A Early lessons from sequencing the Desulfitobacterium pangenome, from carbon to silicon and back again Thomas Kruse*1, Julien Maillard2, Christof Holliger2, Nico Boon3, Jan Gerritse4, Ute Lechner5, Lynne Goodwin6, Patrick Chain7, Betty Lobos8, Willem de Vos1, Hauke Smidt1 1Wageningen University, Laboratory of Microbiology, Netherlands, 2Ecole Polytechnique Fédérale de Lausanne / Laboratory for Environmental Biotechnology, Switzerland, 3Ghent University, Laboratory of Microbial Ecology and Technology (LabMET), Belgium, 4Deltares, Netherlands, 5Martin-Luther-Universität Halle, Institute of Biology/Microbiology, Germany, 6DOE Joint Genome Institute, USA, 7Los Alamos National Laboratory / Bioscience Division, USA, 8Washington University School of Medicine, Genome Sequencing Center, USA

Desulfitobacteria are low G+C Gram-positive, strictly anaerobic bacteria that seem to be ubiquitous in anoxic environments. They have been isolated from environments as diverse as sediment, aquifers, and the human gut. Most of currently available isolates are able to grow by organohalide respiration of one or more halogenated aliphatic and/or aromatic compounds, making them interesting candidates for use in bioremediation of subsurface environments. Previous research on this genus has focused on function and regulation of reductive dehalogenases, and less on other aspects of their metabolism and evolution. Full genome sequences are a pivotal basis for achieving a better understanding of the ecology and evolution of this genus, which may help us to unlock their full potential for bioremediation. The genome sequences of two strains, D. hafniense Y51 and D. hafniense DCB-2, have been available for some years. Recently we have initiated the sequencing of ten additional strains from this genus, bringing the number of sequenced strains to a total of twelve, covering the full taxonomic and physiological width of this genus. Preliminary data analysis revealed genome sizes varying from 3.2 to 5.7 Mbp for D. metallireducens and D. hafniense Y51, respectively. The number of reductive dehalogenase-encoding gene clusters varies from 0 to 7 for D. hafniense DP7 and D hafniense DCB-2, respectively, confirming that desulfitobacteria are less specialised in organohalide respiration than Dehalococcoides spp. and Dehalobacter spp. Combining analysis of the genome data with wet lab experiments provided the necessary basis to propose a refined model for the electron transport chain from the electron donor formate to 3-chloro-4-hydroxyphenyl acetate in the type species D. dehalogenans, consisting of two proton-translocating steps, where hydrogen is produced intracellularly, diffuse out of the cell before being taken up again by an uptake hydrogenase. The availability of twelve Desulfitobacterium spp. genomes can be expected to boost our understanding of the evolution, metabolism and adaptation to the environment of this genus.

484A Peptidolytic systems of Synergistetes bacteria, with an emphasis on a novel fluoroacetate-degrading bacterium, Synergistes sp. strain MFA1 Lex Leong*1, Stuart Denman1, Carl Davis1, Phil Hugenholtz2, Chris McSweeney1 1CSIRO Livestock Industries, Australia, 2Australian Centre for Ecogenomics, Australia

Bacterial species belonging to the Synergistetes phylum can be isolated from diverse environments, and are all characterised as amino-acid fermenters. Synergistetes species use peptides and amino acids for energy production through the reduction of organic or inorganic compounds. This is reflected in their genome content, with an increased presence of amino acid metabolism genes compared to other bacterial species.

Synergistes sp. strain MFA1, a member of the Synergistetes phylum, was recently isolated from the cattle rumen, and degrades fluoroacetate by a novel mechanism of halorespiration. The aim of this study was to explore effects of peptides on fluoroacetate degradation by strain MFA1, and examine the diversity of the peptidolytic systems of the eleven sequenced bacterial genomes from the Synergistetes phylum.

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Strain MFA1 showed greater growth rates when cultured on peptide sources such as tryptone and yeast extract compared to free amino acid mixtures like casamino acids or synthetic amino acid mixes. Similarly, the degradation of fluoroacetate by strain MFA1 was enhanced when using peptide sources compared to free amino acids. Amino acid profiling of the cultures supplemented with fluoroacetate showed a wider range of utilized amino acids compared to without fluoroacetate addition. Amino acids such as glutamate, glycine, histidine, arginine, asparagine, serine, threonine and lysine were observed to be degraded to a greater extent in the presence of fluoroacetate. Strain MFA1 did not degrade branched-chain amino acids under any condition. HPLC chromatogram showed reduction of specific peptide peaks within the cultures, and MALDI-TOF/TOF MS/MS analysis identified oligopeptides consisting of hepta-glutamate residues at the N-terminal.

The draft genome of strain MFA1 was found to contain the entire complement of genes involved in the amino acid metabolism pathways for those identified from the amino acid profiling studies. In addition, a diverse set of 19 genes encoding peptidases and four operons of ATP-binding cassette oligopeptide/dipeptide transporters. Phylogenomic comparison of strain MFA1 with published genomes from eleven other Synergistetes species showed that genes encoding the peptidases pepM, pepF, ggt, pepP, lap, dpep1 and opp/dpp operons are all conserved across the phylum. But some peptidases are more exclusive to certain individuals, such as pip and pepO, which were unique to strain MFA1 and other species in family Synergistaceae, except for the thermophilic Thermanaerovibrio acidaminovorans.

These analyses demonstrate that peptidases play an essential role in nutritional and other cellular functions in bacterial species from the phylum Synergistetes. Peptides are an important source of energy for the growth of strain MFA1, and from the increase growth, peptides rich with glutamate may also enhance fluoroacetate degradation.

485A Metaproteomic study of functional microbial community in thermophilic anaerobic digestion Yi-Wen Lin, Yi -Chia Chang, Shir-Ly Huang* National Central University, Taiwan

Biogas, a mixture of methane and carbon dioxide, is a combustible and renewable source of energy. Because of the high cost of fossil fuels, the carbon emission and global warming issues, throughout the world, scientists are actively looking for alternative energy sources. Biogas is generated as a co-product of the waste treatment process called anaerobic digestion. However, the biological system and its bio-control inside of the digester are still relatively unknown. This study is anticipated to use metaproteomic approach and clone library of 16S rRNA gene to investigate the microbial community from thermophilic anaerobic digesters. The digester contains pig manure for the biogas production at 55°C. A total of over three hundreds of proteins were identified by mass spectrometry from the extracted metaproteome. The sources of the identified proteins were mainly from eubacteria. They were Proteobacteria (about 60%), Firmicutes, and Acidobacteria contributing to the hydrolysis of complicated biopolymers and acidogenesis to produce biogas. Compare to other investigations which used 16S rRNA gene analysis, Bacteroidetes was dominant in 15°C and 33°C anerobic digesters. Firmicutes were relatively dominated in the thermophilic and hyperthermophilic anaerobic digesters. Our results showed that Proteobacteria constitute more than 60 percent of bacteria in 55°C and differed from the other investigations which using 16S rRNA gene analysis. Since the substrate applied in the present study was different from other studies, it contributes the different microbial community. The swine manure which used in our anaerobic digester as sole supply is a complex substrate due to the complicated composition of feed to swine growth. The diversity and abundance of bacteria in anaerobic digestion, found by this study using metaproteomic approach and 16S rRNA gene approach, could be explained by the input of different and complicated substrate sources and the ability of the microbial community to degrade a wide range of macromolecules. For the methanogenesis stage, within the methanogenic archaea, Methanocaldococus jannaschii, Methanothermobacter thermoautotrophicum, Methanothermus fervidus, Methanosarcina mazei, Methanosarcina sp., and Methanosarcina barkeri, were detected by metaproteomic approach. Methanothermobacter sp., Methanobacterium formicicum, and Methanosarcina thermophila were detected by 16S rRNA gene sequences. Enzymes involved in methanogenesis were also detected from the methanogens. Acetyl-CoA decarbonylase which was detected from Methanosarcina acetivorens produces methane from acetate. Methyl-coenzyme M reductase from M. thermoautotrophicum, M. fervidus, and Methanosarcina barkeri catalyzes the reduction of

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methylcoenzyme M to methane. However, the observed low archaeal diversity might be due to the absence of archaeal protein sequences in the database. 16S rRNA gene cloned libraries of the methanogens was also included to strengthen the information derived from metaproteomic approach. The present study is the first report on the investigation of metaproteome in a thermophilic anaerobic digester. It also provides information of the functional microbial communities using swine manure as sole feedstock for the biosynthesis of biogas.

486A A protein exclusive of filamentous cyanobacteria involved in determination of cell division plane Dinka Mandakovic*1, Carla Trigo1, José Enrique Frías2, Enrique Flores2, Luz Mónica Vásquez1 1Pontificia Universidad Católica de Chile, Chile, 2Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Espana

Multicellular filamentous cyanobacteria are organisms that differentiate cells along the filament with specialized functions, including heterocysts that fix nitrogen. In the genomic comparative study done by Stucken and coworkers (2010), 10 genes were identified as exclusive of filamentous cyanobacteria. One gene of this group is Anabaena sp. PCC 7120 all2320, which encodes a protein of unknown function that bears one transmembrane segment and one coiled coil domain, and therefore could have a membrane-associated function in filamentous cyanobacteria. The aim of this work was to characterize this protein, determine its localization and uncover its function in filamentous cianobacteria. Analyzing gene expression in Anabaena sp. PCC 7120 by RT-qPCR, neither induction nor repression was observed for all2320 in the presence or absence of combined nitrogen in the culture media until 72 hours of growth. All 2320 gene expression was also observed by Northern Blot analysis using RNA isolated from cultures of Anabaena sp. grown with ammonium and incubated in the absence of combined nitrogen for several periods of time up to 24 hours. A double-stranded gene probe of all2320 hybridized to an RNA molecule of about 2.0 kb, which probably corresponds to a transcript covering only all2320 and therefore evidencing that this gene is monocistronic. No induction was observed during the N step-down time-course for all2320 transcript. To study protein localization, All2320 was expressed in Escherichia coli and purified, and an anti-All2320 polyclonal antibody was generated. Using immunofluorescence labeling and confocal microscopy, we could observe that All2320 localizes at one cell pole during cell division, location that seems to arise before the Z-ring is formed. To get a better insight about the function of All2320, mutant strains bearing inactivated copies of all2320 were generated. Although not fully segregated, a clear phenotype was observed in the mutant strain. By light microscopy we could observe a distorted cell division, with production of cells showing division in more than one plane, forming cluster-like structures. This mutant strain was also visualized by transmission electron microscopy, and besides the expected altered division planes, a drastic ultrastructure alteration was noticed. Mutants contained less carboxysomes that were larger than in the wildtype. Our results suggest a function of All2320 during cell division, related to filament growth.

487A Genomic investigation of the filamentous foam forming Candidatus 'Microthrix parvicella' isolated from activated sludge Simon McIlroy*1, Rikke Kristiansen1, Mads Albertsen1, Søren Karst1, Simona Rossetti2, Valter Tandoi2, Jeppe Nielsen1, Steve Petrovski3, Robert Seviour3, Per Nielsen1 1Aalborg University, Denmark, 2Water Research Institute, CNR, Italy, 3La Trobe University, Australia

"Microthrix parvicella" is a Gram positive, filamentous member of the Actinobacteria. It is observed in activated sludge wastewater treatment plants where it is often associated with the poor sludge separation problems known as 'bulking' and 'foaming'. Despite its importance little detail is known or certain about its physiology and why it thrives in activated sludge wastewater treatment plants; an improved understanding of this organism is likely to provide the key to improving future control strategies.

In the present study the genome sequence of "Microthrix parvicella" strain RN1 was obtained and annotations made for the key metabolic pathways, entailing the carbon, nitrogen and phosphorus metabolism; to identify possible metabolic strategies for survival under the dynamic conditions of activated sludge treatment. The genome sequence was also compared to related genome fragments

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in deep metagenomes, generated for full-scale wastewater treatment plants, thought to represent in situ genotypes.

In situ data suggest that "Microthrix" is a lipid specialist, with the ability to take up and store large amounts of lipid material, and thriving in plants with a high influent content. This was confirmed by the analysis of its genome where multiple copies of the key enzymes involved in both the β-oxidation of long chain fatty acids, and the triacylglycerol synthesis/utilisation pathways where located, along with both intracellular and extracellular lipases. The key enzymes for the production of polyphosphate, but not glycogen or PHA, were also located. In enhanced biological phosphorus removal (EBPR) configured systems, the ability of "Microthrix" to accumulate lipids in the carbon rich anaerobic phase, for use in the subsequent carbon deficient aerobic phase, is considered key to its success. It is not clear from the genome where the energy for this is sourced, although polyphosphate hydrolysis and release may contribute. The genome contained enzymes for the complete TCA cycle; and although the key enzymes for the glyoxylate shunt were absent, the alternative ethylmalonyl-CoA pathway appears to explain its ability to grow on C2 compounds (from intracellular β-oxidation of long chain fatty acids). The presence of nitrate and nitrite reductases indicate an ability to denitrify to nitric oxide, although only nitrate reduction has been observed in pure culture and in situ.

488A Changes in the metabolic profile of Streptomyces coelicolor under heavy metal ion stress Anne Morgenstern*1, Anne Behrend2, Dieter Spiteller3 1Max-Planck Institute For Chemical Ecology, Germany, 2Leibniz-Institute of Vegetable and Ornamental Crops, Germany, 3University of Konstanz, Germany

Actinomycetes, especially Streptomyces sp. are well known to produce a wide variety of secondary metabolites. The production of these compounds is dependent on environmental conditions. Biotic factors, such as neighboring microorganisms, and abiotic factors, for example heavy metal ions, have a strong impact on the metabolite production triggering cryptic natural product biosynthesis in microorganisms.

The model organism Streptomyces coelicolor produces several secondary metabolites in response to different biotic and abiotic factors, among them the pigments actinorhodin and undecylprodigiosin. It was shown in previous studies that the production of actinorhodin is induced by rare earth element ions, in particular scandium ions. The prodigiosin biosynthesis is known to be strongly upregulated upon salt stress. However, so far never a strong increase of both pigments under the same stress conditions was observed.

Here we report the simultaneous appearance of various different phenotypes of Streptomyces coelicolor M145 grown on soy flour medium (SFM) supplemented with cobalt ions or other bivalent heavy metal ions. While on SFM medium S. coelicolor grows as brown-grey colonies, on SFM medium supplemented with 70 mM Co2+ ions a variety of strongly pigmented phenotypes was observed. In order to analyze this adaptation to heavy metal ion stress red and blue colonies were isolated and further cultivated under Co2+-stress. LC-MS and GC-MS analysis revealed drastic differences in the secondary metabolite profiles of the wild type, the red and the blue phenotype.

The metabolic profile of the red phenotype, which we analyzed in some detail so far, revealed the overproduction of undecylpyrrol, undecylprodigiosin, streptorubin B and some unknown pigments. For further structure elucidation these pigments were extracted with methanol from the cells and purified by LH20 and RP18 chromatography. Currently the novel secondary metabolites are characterized by high resolution mass spectrometry, tandem mass spectrometry and NMR.

489A A new way of activating non-substituted aromatic hydrocarbons: Identification of direct carboxylation of naphthalene Housna Mouttaki*, Jörg Johannes, Rainer U. Meckenstock Institute of Groundwater Ecology, Helmholtz Zentrum München, Germany

The understanding of biochemical processes underlying the anaerobic degradation of non-substituted aromatic compounds are still at their infancy. Metabolite analyses have indicated that for the model compound naphthalene, carboxylation rather than methylation was the first reaction activating such

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chemically stable molecule. We have recently brought biochemical evidence confirming the carboxylation reaction in the sulfate reducing culture N47. Naphthalene carboxylase is biotin-, and ATP- independent. Oxygen and strong reducing agents such as Ti(III) citrate or sodium dithionite strongly inhibit the enzyme activity. Divalent metal ions such as Mg2+ or Mn2+ have a slight inhibitory effect while ZnCl2 at concentrations of 5mM completely inhibited the enzyme activity. Naphthalene carboxylase exhibits an isotopic exchange capability with a Vmax of 2.9 pmol.sec-1 and a Km of 9 nmol indicating the formation of free reversible intermediates. Furthermore, the enzyme is reversible and catabolyzes the formation of naphthalene from 2-naphthoic acid in the presence of only N47 raw extract. The decarboxylation reaction has a specific activity of 17 pmol min-1 mg-1 of protein. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.

490A The role of dehydroascorbate as surveillance molecule of oxidative stress and programmed cell death Omer Murik*, Aaron Kaplan Hebrew University of Jerusalem, Israel

Although apoptosis is a very well-studied mechanism of programmed cell death (PCD) in metazoans, the roles and mechanisms of PCD in plants and microorganisms is still poorly understood. We are studying the PCD machinery of the model green algae Chlamydomonas reinhardtii, which is induced by oxidative stress. Paradoxically, C. reinhardtii oxidative stress-induced cells are more sensitive to H2O2 treatment although they possess higher antioxidants activities. Dehydroascorbate, the oxidized product of ascorbate peroxidase (APX), level was higher in conditions favoring PCD and was able to induce PCD. The role of cytochrome c release from the mitochondria in this PCD, a hallmark of metazoans apoptosis, was demonstrated using a Complex III lacking mutant showing differential response to H2O2 and DHA induced PCD. Finally, the expression and activity of metacaspases, the executioners of paoptosis, was shown to be associated with C. reinhardtii PCD. The function of PCD in ecology and evolution of phytoplankton will be discussed.

491A Insight into the sulfur metabolism of Thioalkalivibrio sulfidiphilus HL-EbGr7 with quantitative proteomics Gerard Muyzer*1, Dimitry Yu. Sorokin2, Peter Schaap3, Peter Verhaert4, Martijn Pinkse4 1Institute for Biodiversity and Ecosystem Dynamics, Netherlands, 2Winogradsky Institute for Microbiology, Russia, 3Wageningen University, Netherlands, 4Delft University of Technology, Netherlands

Thioalkalivibrio sulfidiphilus HL-EbGr7 is an obligately chemolithoautotrophic sulfur-oxidizing bacterium using CO2 as a carbon source and reduced inorganic sulfur compounds as an energy source. Tv. sulfidiphilus was isolated as a dominant sulfide-oxidizing bacterium from a full-scale bioreactor removing H2S from biogas (Sorokin et al., 2012). Recently, the genome of Tv. sulfidiphilus was sequenced (Muyzer et al., 2011). The genome consists of a single circular chromosome with a size of 3.46 Mbp, and has 3366 genes. Different genes encoding enzymes that are involved in the sulfur oxidation were detected, such as fccAB, soxABXYZ, drsABEFHCMKLJOPNR, sorA, aprBA and sat. In addition, we found genes encoding the heterodisulfide reductase complex, which was highly similar to that found in Acidithiobacillus ferroooxidans, in which it was hypothesized to be involved in the sulfur metabolism (Quatrini et al., 2009). To determine the role of these enzymes in the sulfur metabolism, we grew Tv. sulfidiphilus in chemostats with different sulfur compounds and under different redox conditions. Subsequently, we toke samples for quantitative proteomics to look at gene expression. Here we will present the results of this study.

492A Benzene degradation under anaerobic conditions Kerstin Nicolaisen*, Rainer U. Meckenstock Institute of Groundwater Ecology, Helmholtz Zentrum München, Germany

The BTEX compounds (benzene, toluene, ethylbenzene and xylene) are very toxic and highly water solubility. Benzene is the most difficult to degrade, since its aromatic ring is hardly accessible. Under oxic conditions BTEX can be easily biodegraded by well-known oxygenase pathways. In contrast, in aquifer systems where anoxic conditions dominate, novel degradation reactions have to take place.

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Until now three initial activation reactions for anaerobic benzene degradation are postulated: methylation, hydroxylation and carboxylation. In order to highlight which reaction actually takes place we are analyzing the iron-reducing culture BF and the sulphate-reducing culture BPL.

A proteomic dataset of strain BF was generated by LC/ESI-MS/MS analysis of proteins separated by 1D-gel electrophoresis. This led to the identification of a putative anaerobic benzene carboxylase (Abc) consisting of several subunits. Two show 43% and 37% sequence identity to phenylphosphate carboxylase subunit PpcA and PpcD from A. aromaticum strain EbN1. By shot gun sequencing of the BF genome by 454 pyrosequencing also the corresponding genes could be identified. They are encoded on the same operon as benzoylCoA-Ligase. Growth analysis of strain BPL showed that phenol and toluene, which would be intermediates during hydroxylation respectively methylation, are not used as substrates by this strain. Proteomic analysis of benzene grown cells did also not reveal putative enzymes for these reactions. During GC-MS metabolite analysis with stable isotope-labelled benzene or bicarbonate buffer benzoate could be detected.

Based on the proteomic and genomic data from strains BF and BPL, carboxylation is considered to be the first step in anaerobic benzene degradation.

493A Study of a novel oxygen-sensitive, denitrifying Bacillus abundant in soil Maja Nielsen*, Vincenzo Cerciello, Kasper U. Kjeldsen, Eline P. Hansen, Lars Schreiber, Andreas Schramm, Kai Finster Aarhus University, Denmark

Denitrifying microorganisms are typically characterized as facultative anaerobes that grow best by oxygen respiration but start reducing nitrate to N2 when oxygen becomes limiting. The onset of denitrification is usually tightly regulated by oxygen and N oxide concentrations, which affect both the level of gene expression and the activity of the N-oxide reductases. Most information about the biochemistry and regulation of denitrification is derived from a few Gram-negative model strains e.g. Paracoccus denitrificans, while little is known about Gram-positive denitrifiers; in fact, denitrification has only recently been recognized as a widely distributed property of the Gram-positive genus Bacillus.

Here we report the isolation and characterization of a novel denitrifying Bacillus strain abundant in soil, which apparently is unable to grow with oxygen as electron acceptor.

The strain MEV2011 was isolated on complex medium under anoxic, denitrifying conditions from the highest dilution of a soil sample; its high natural abundance was subsequently confirmed by MPN-PCR targeting the nitrous oxide reductase (nosZ) gene specific for the strain. The closest described relative of strain MEV2011was the complete denitrifier Bacillus azotoformans (97% 16S rRNA gene similarity). Surprisingly, strain MEV2011 was unable to grow in the presence of oxygen. However, online microsensor measurements of oxygen in liquid medium showed that strain MEV2011 removed atmospheric concentrations of oxygen and started to grow by denitrification upon oxygen depletion. The lag phase was dependant on the initial oxygen concentration. Draft genome analysis of strain MEV2011 revealed the presence of genes encoding typical oxygen protection enzymes (catalase, superoxide dismutase, peroxidase) and at least one terminal oxidase. It remains therefore enigmatic why oxygen respiration cannot be coupled to growth in the novel Bacillus strain. In contrast, the onset of denitrification only after complete oxygen removal may be explained by the canonical suppression of denitrification gene expression by oxygen. This hypothesis is currently tested by transcript analyses.

In conclusion, the novel Bacillus strain MEV2011 behaves like an obligate denitrifier, unable to grow with oxygen, despite its ability to remove oxygen at atmospheric concentration.

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494A Oxidation kinetics of nitrite-oxidizing bacteria from activated sludge Boris Nowka*1, Holger Daims2, Eva Spieck1 1University of Hamburg/Microbiology, Germany, 2University of Vienna/Microbial Ecology, Austria

Aerobic nitrite oxidation is the second step of nitrification, a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing microorganisms. Nitrite-oxidizing bacteria (NOB), consist of five genera, of which Nitrobacter, Nitrospira and the recently discovered Nitrotoga were detected in activated sludge of wastewater treatment plants. Since these slow growing, chemolithoautotrophic bacteria use nitrite as their sole energy source, growth is directly linked to the availability of this substrate and the kinetics of its oxidation. Interestingly, all three genera occur in the same municipal wastewater treatment plant, which leads to the hypothesis of different niches in this habitat. Additionally, also members of two different sublineages of the predominant Nitrospira could be found in the same WWTP, suggesting niche separation even within the same genus.

The aim of this study was an advanced comparative analysis of NOB oxidation kinetics and growth characteristics with regard to substrate-dependent niche partitioning among nitrite-oxidizers. Therefore, we compared pure cultures of NOB isolated from activated sludge and further non-marine habitats like soil and heating water. The measurements of nitrite-dependent oxygen consumption were carried out directly in pure cultures by a standardized protocol and a precise microsensor-based respiration system.

The measurements revealed different oxidation kinetics between genera, but also between species of the same genus according to their habitat. Members of the genus Nitrobacter (r-strategists) showed high nitrite turnover rates (Vmax) of 61 (Nb. hamburgensis; soil), 81 (Nb. winogradskyi; soil) and 179 µmol N/mg protein/h (Nb. vulgaris; activated sludge). In contrast, members of the genus Nitrospira (K-strategists) have lower maximum nitrite turnover rates under the applied conditions. Here, values of 48 (Nitrospira defluvii; activated sludge) and 22 µmol N/mg protein/h (Ns. moscoviensis; heating water) were obtained. Furthermore, the results confirmed great differences in the half-saturation constants with a sixfold higher affinity for nitrite of Ns. defluvii (KS 11 µM) compared to Nb. vulgaris (KS 66 µM), which could be an indicator for the prevalence of Nitrospira during wastewater treatment. Additionally, a wide range of different KS-values were measured for the soil-derived nitrite oxidizers Nb. hamburgensis (KS 441 µM), Nb. winogradskyi (KS 365 µM) and the heating water-derived Ns. moscoviensis (KS 7 µM). It can be speculated that the KS-values reflect the specific availability of nitrite in the corresponding environment. Together with other growth characteristics these measurements give an extended view on nitrite-oxidizing bacteria and factors controlling their distribution in nature.

495A The mechanism of heterocyst development in the cyanobacterium Anabaena sp. PCC7120 under nitrogen deprived conditions Masayuki Ohmori*, Shigeki Ehira Chuo University, Japan

Heterocysts are terminally differentiated cells of filamentous cyanobacteria specialized for nitrogen fixation. Upon limitation of combined nitrogen in the medium, particular vegetative cells differentiate into heterocysts with a regular spacing of 10 to 15 cells. Differentiating cells undergo drastic metabolic and morphological change, namely, O2-evolving photosystem II is inactivated, a rate of respiration is increased, and a thick envelope is formed outside of the cell wall, which limits oxygen diffusion into the cells. Because heterocysts are unable to fix CO2 photosynthetically, vegetative cells supply carbohydrate to heterocysts, and, in return, vegetative cells receive nitrogen fixation products from heterocysts. Cyanobacteria store carbon assimilated by photosynthesis in the form of glycogen, which is catabolized to provide reductants and ATP required for nitrogen fixation. Thus, carbohydrate metabolism is very important for nitrogen fixation in the filamentous cyanobacteria.

Real time quantitative reverse transcription-PCR and gel mobility shift assay were used to determine gene expression and interaction of NrrA with the promoter region, respectively. The glycogen content of whole filaments was determined according to the method described by Forchhammer and Tandeau de Marsac. The activity of nitrogen fixation was determined by acetylene reduction method.

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We have found that a nitrogen-regulated response regulator, NrrA, which is involved in heterocyst differentiation, controls glycogen catabolism. The transcript levels of genes involved in glycogen catabolism were decreased by disrupting the nrrA gene. Glycogen accumulation and depression of nitrogenase activity were observed in this disruptant. It was shown that NrrA bound to the promoter region of the glgP1 gene for glycogen phosphorylase and enhanced its expression. It is also found that NrrA regulates the expression of sigE gene, encoding a group 2 sigma factor of RNA polymerase. SigE was shown to be a transcriptional activator of the xfp operon, encoding enzymes of glycolysis and the pentose phosphate pathway. It is concluded that NrrA controls not only heterocyst differentiation but also glycogen catabolism in Anabaena sp. strain PCC 7120.

496A Meta-genomic analysis for the elucidation of physiology of an anammox bacterium, Candidatus 'Brocadia sinica' Mamoru Oshiki*1, Kaori Shinyako2, Hisashi Satoh1, Satoshi Okabe1 1Hokkaido University, Japan, 2Tokyo Engineering Consultants Co. LTD, Japan

Anaerobic ammonium oxidation (anammox) bacteria have been known as one of the fastidious microorganisms. Until now, no pure isolate of anammox bacteria has been obtained, which make difficult to understand their life even in laboratories. Genome information will shed light on the nature of anammox bacteria because the genome is blueprint of life. In the present study, the authors performed meta-genomic analysis to an anammox bacterium, Candidatus ‘Brocadia sinica’ in order to obtain their genetic blueprint.

Ca. ‘B. sinica’ was cultivated in an up-flow column reactor operated with inorganic nutrient medium at 38°C. Phylogenetic affiliation of Ca. ‘B. sinica’ was checked by the direct sequencing of the nucleotide sequences spanning 16S to 23S rRNA gene and their abundance was quantified by fluorescence in-situ hybridization (FISH) analysis. For the meta-genomic analysis, nucleic acids were extracted and subjected to 454-pyrosequencing analysis with GS FLX (Roche, BSU, Switzerland). Read sequences were assembled with GS de novo assembler 2.3 and the gene annotation was performed with CRITICA and glimmer 2.

The enriched culture of Ca. ‘B. sinica’ exhibited extremely high nitrogen removal rate, 32kgN m-3 d-1, and Ca. ‘B. sinica’ accounted for above 90% in the total biomass. This outcome indicated that the prepared biomass was suitable for the subsequent meta-genomic analysis and may allow us to obtain the whole genome sequence of Ca. ‘B. sinica’. 454 pyrosequencing produced 1,204,328 reads with an average length of 336 bps, which assembled into the fifteen large fragments, fourteen contigs and single scaffold. Total size of these fifteen fragments was 4.00 Mbps with GC contents of 41% and 3,900 genes were annotated. The completeness of the fifteen fragments as the genome sequence of Ca. ‘B. sinica’ was estimated based on the presence of core gene sets, which indicated that at least 97% of genome sequence was covered by those sequences. This outcome indicated that the authors obtained almost complete genome sequence of Ca. ‘B. sinica’. The obtained genome information allowed us to characterize the metabolic capacities of Ca. ‘B. sinica’ and the comparative genomic analysis with the one of another anammox bacterium, Ca. ‘Kuenenia stuttgartiensis’ provided novel insights as for the difference in their physiology. The details of our current achievements will be introduced in my presentation. The present study demonstrates the usefulness of the meta-genomic approach to investigate the metabolic status of fastidious microorganisms.

497A A bacterium representing marine agarolytic novel genus in the family Flavobacteriaceae, isolated from seawater Seong Chan Park*1, Yeoung Min Hwang1, Han Na Choe1, Keun Sik Baik1, Ye-Seul Hwang2, Duwoon Kim2, Chi Nam Seong1 1Sunchon National University, South Korea, 2Chonnam National University, South Korea

Two yellow colored, rod-shaped, Gram-reaction-negative and agarolytic bacterial strains, designated KYW560T and KYW563T, were isolated from a seawater sample of Gwangyang Bay, Korea. Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strains KYW560T and KYW563T shared 96.8 % sequence similarity and formed a robust monophyletic group within the radiation of the family Flavobacteriaceae. The distinct clade with varying branching positions depending on the tree inferring algorithm reflects a novel generic status within the family Flavobacteraceae. The primary isoprenoid quinone as MK-6 (menaquinone-6) and the DNA G+C

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content (34.3-35.0 mol%) were consistent with its assignment to the family Flavobacteriaceae, but the polar lipid profile, fatty acid composition and overall phenotypic traits demonstrated that the test strains were not closely affiliated to any of the previously described genera. The data obtained from these polyphasic taxonomic studies revealed that the two agarolytic isolates could be classified as representatives of two novel species in a new genus, Gwangyangia gen. nov., with Gwangyangia aliagarivorans sp. nov. [type strain is KYW560T (=KCTC 23855T)] as the type species and Gwangyangia aliagarivorans sp. nov. [type strain is KYW563T (=KCTC 23857T)] as a second species.

498A Constraining the nitrogen metabolism of deep-sea vent chemosynthetic microbes using stable nitrogen isotopic analyses: An experimental study at in situ seafloor pressures Ileana Perez-Rodriguez*1, Stefan Sievert2, Marilyn Fogel1, Dionysis Foustoukos1 1Carnegie Institution of Washington, USA, 2Woods Hole Oceanographic Institution, USA

At deep-sea hydrothermal vents subsea floor mixing of oxygenated cold seawater and high-temperature reducing vent fluids induce sharp thermal and chemical gradients across the mixing boundary allowing chemosynthetic microorganisms to mediate carbon and energy transfer from the geothermal source to higher trophic levels. In the last few years, it has become evident that the physico-chemical conditions at near-seafloor hydrothermal sites reflect the potential importance of nitrate as a key node in the network of nitrogen pathways for deep-sea microorganisms. However, the role of deep seawater NO3

- has not been fully explored, despite the highly energetic nature of the microbially mediated nitrate reduction reactions. Because chemosynthetic Epsilonproteobacteria are increasingly recognized as an ecologically relevant group at deep-sea vents, we are using Caminibacter mediatlanticus, a nitrate-reducing epsilonproteobacterium, as a model system. To this end, we are assessing the assimilatory and dissimilatory pathways involved in nitrogen metabolism at pressure conditions similar to those encountered in deep-sea vent environments. Through a constrained investigation of the nitrogen stable isotope exchange between the dissolved nitrogen species (NO3

-, NH4+) and the nitrogen assimilated into biomass, we are researching the chemical

signatures of nitrogen-based metabolic processes during C. mediatlanticus growth.

In detail, measurements of 15N/14N fractionation between NO3- and NH4

+ obtained during C. mediatlanticus growth under different hydrostatic pressure regimes have been obtained together with growth rates and rates of dissimilatory nitrate reduction to ammonium (DNRA). At the same time, incubations using a series of 15N-labeled analyses starting with 15N-labeled NaNO3 and NH4Cl have been made to track the incorporation of nitrogen into the microorganism's biomass during growth. Our data includes the successful establishment and sampling of batch cultures growing under hydrostatic pressures of 50 and 200 bar, utilizing a flexible gold/titanium reaction cell. This batch reactor allows for samples to be retrieved during the course of experiments, and thus, to monitor growth at in-situ seafloor pressures. Results indicate that higher hydrostatic pressure conditions (50 bars and 200 bar) significantly decrease the microorganism's growth rates, cell biomass and rates of catalyzed nitrate reduction reactions. Moreover, 15N-labeled experiments suggest that there is a link between dissimilatory and assimilatory pathways during C. mediatlanticus growth, with a fraction of the ammonium produced during respiration becoming incorporated into biomass. These data help to improve the understanding of nitrogen metabolism in anaerobic chemosynthetic nitrate reducing microorganisms at deep-sea hydrothermal vents, and provide new proxies for estimating the importance of this process for biomass production in the subseafloor at deep-sea vents.

499A Relative importance of chemosynthesis in a polluted tropical estuary Alex Prast*, Camila Signori, Giselle Parno, Bruno Rego, Ricardo Pollery, Angela Sanseverino University Federal of Rio de Janeiro, Brazil

The aim of this study was to evaluate the relative importance of chemosynthesis in relation to bacterial production at Guanabara Bay (Rio de Janeiro, Brazil), a polluted and eutrophic tropical estuary. This study is part of the long term ecological research (LTER) called PELD-Guanabara and aims a better understanding of the relative importance of the microbial processes on the carbon cycle. Chemosynthetic and bacterial production rates were determined by 14C and 3H-leucine incorporation and incubations in the dark. Measurements were performed in two different areas: A, deeper, highly influenced by the sea and with less human influence, and D, shallower, with lower water circulation and with higher human impact. Higher rates of dark carbon fixation and bacterial production were observed at polluted area. Higher rates of dark carbon fixation and bacterial production were observed

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at the most impacted area. However, the relative importance of chemosynthesis compared to the bacterial production varied during the year. It was verified a negative correlation between transparency and microbial processes, as well as between dissolved oxygen and bacterial production. This process was also influenced by temperature and dissolved organic carbon concentrations.

500A Different ecophysiologies of two Dehalococcoides isolates from the Bitterfeld region of germany Marlén Pöritz*1, Ulrike Heinemann1, Lorenz Adrian2, Tesfaye Wubet3, Mika Tarkka3, Ivonne Nijenhuis2, Ute Lechner1 1Martin-Luther-University Halle-Wittenberg, Institute of Biology/Microbiology, Germany, 2Helmholtz-Centre for Environmental Research – UFZ, Department of Isotope Biogeochemistry, Germany, 3Helmholtz-Centre for Environmental Research – UFZ, Department of Soil Ecology, Germany

Soils, groundwater and sediments of the Bitterfeld area in central Germany are highly contaminated by different chlorinated compounds as a result of discharges from the chemical industry. The contaminants range from chlorinated aliphatics, hexachlorocyclohexane and chlorobenzenes to chlorinated dibenzo-p-dioxins and dibenzofurans. Recently, two Dehalococcoides strains were enriched from this area. Strain DCMB5 was isolated from highly dioxin-contaminated river sediment. Strain BTF08 was enriched from a groundwater well contaminated by chlorinated ethenes. The aim of this study was to determine the relatedness and distinctness of both strains at the genomic and metabolic levels to select specific properties as targets for a contaminant-specific monitoring of the in situ dehalogenation capacity.

Whole genome sequencing was performed on total DNA from both strains using 454 pyrosequencing. After gap-closure the genomes were automatically annotated and genes encoding proteins homologous to reductive dehalogenases (Rdh) were identified and compared with those of strains CBDB1, VS, 195 and FL2. The strains were cultivated under strictly anaerobic conditions in mineral medium with acetate and CO2 as carbon sources, hydrogen as electron donor and different chlorinated compounds (25-500 µM) as electron acceptors for organohalide respiration. Dehalogenation of the chlorinated compounds was monitored by gas chromatography or high performance liquid chromatography.

Comparison of the genomes of both strains revealed a conserved core with some marked differences such as the presence of three copies of a transposable element in strain BTF08 and of a CRISPR locus in strain DCMB5. The rdhA genes encoding the catalytic subunit of Rdhs are mostly located in two so-called high plasticity regions. The genomes of strains DCMB5 and BTF08 contained 23 and 20 non-identical rdhA genes, respectively. Only 8 rdhA genes from both strains were orthologues. Nine rdhA genes of strain DCMB5 and one rdhA gene of strain BTF08 were unique with no orthologues in other cultivated Dehalococcoides strains. Both strains were able to dehalogenate hexa-, penta-, 1,2,3,4- and 1,2,3,5-tetrachlorobenzene, 1,2,3,4-tetrachloro- and 1,2,3-trichlorodibenzo-p-dioxin. However, strain DCMB5 additionally dechlorinated 1,2,3-trichlorobenzene, 1,2,4-trichloro- and 1,3-dichlorodibenzo-p-dioxin, and different tetra- and trichlorinated phenols and was able to couple most of these activities to growth. In contrast, strain BTF08 showed a stronger preference for chlorinated aliphatics as electron acceptors for organohalide respiration. The different physiologies of both strains correlate with the presence of specific rdhA genes. The presence of a gene in strain DCMB5 encoding an orthologue of CbrA, the functionally characterized chlorobenzene Rdh of strain CBDB1, suggests that cbrA is a suitable biomarker for chlorobenzene- and dioxin-respiring activities. The combination of genes encoding orthologues of TceA and PceA (tri- and tetrachloroethene Rdhs, respectively), which are only found in strain BTF08 also demonstrates that the nature of the prevailing chloroorganic pollutant selected for Dehalococcoides strains with a specific set of rdhA genes.

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501A Genome of Lactococcus piscium strain MKFS47 from modified atmosphere packaged meat Riitta Rahkila*1, Per Johansson1, Margarita Andreevskaya2, Pia Laine2, Lars Paulin2, Petri Auvinen2, Johanna Björkroth1 1Department of Food Hygiene and Environmental Health, University of Helsinki, Finland, 2Institute of Biotechnology, University of Helsinki, Finland

Lactococcus piscium is a psychrotrophic lactic acid bacterium (LAB) that may form a significant part of the bacterial community in late shelf-life, modified atmosphere packaged (MAP) meats or fish. The presence of lactococci in meat has been underestimated, and the characteristics and the spoilage potential of this species are poorly known. We sequenced the complete genome of L. piscium MKFS47 (Project ID: 109289) originating from MAP broiler meat using a Roche 454 Life Sciences GS FLX system (27 x coverage) complemented with a large library of Solexa-Illumina short reads to correct 454 consensus errors. The sequences were assembled into contigs using Newbler, and gaps between contigs were closed by sequencing PCR products. Coding sequences were predicted using a combination of EasyGene and Prodigal, combined with a manual correction. Functional annotation of the gene products was inferred using RAST and PANNZER, followed by a manual correction of disagreements. The genome contains a single circular chromosome of 2 394 138 bp. From the 2290 CDS several genes that might explain the competitiveness of L. piscium in meat environment were identified. The expression of these genes in L. piscium growing in mixed community in meat was studied with RT-qPCR. The results of this study offer novel insights into the adaptation of this LAB to meat niche. Knowledge on the characteristics of spoilage bacteria is needed in ensuring quality and safety of packed meat.

502A Response to phosphorus limitation by the chemoheterotrophic strain Pseudovibrio sp. FO-BEG1 Stefano Romano*, Vladimir Bondarev, Thorsten Dittmar, Heide Schulz-Vogt Max Planck Institute for Marine Microbiology, Germany

Strain FO-BEG1 is an Alphaproteobacterium related to Pseudovibrio denitrificans. It was isolated from a Beggiatoa sp. co-culture and previous genomic and physiological analysis suggests that this strain exhibits a versatile life style. Besides different metabolic abilities, several open reading frames involved in symbiont-host interaction and secondary metabolites production were found.

When strain FO-BEG1 grows in a defined medium under phosphorus limited conditions it produces a so far not characterized yellow compound which is released into the medium. In order to investigate the physiological response and the secondary metabolites production of strain FO-BEG1 under phosphorus limitation, we performed chemical, physiologic, proteomic and exo-metabolomic analyses. The results of the physiological experiments, supported by the proteomic data, demonstrated that under phosphorus-limited conditions strain FO-BEG1 induced the synthesis of phosphoester-bonds degrading enzymes (e.g. alkaline phosphatase) and high-affinity transporters for the uptake of phosphates and phosphonates. Moreover, a drastic change in polar lipids composition was observed, with a substitution of phosphorus containing with phosphorus-free lipids. Interestingly, during the growth in phosphorus-limited conditions we could not detect a complete depletion of the stored polyphosphate. Furthermore, we observed a conspicuous accumulation of polyhydroxyalkanoates in the cells.

The exo-metabolome was analyzed by means of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The analysis revealed that the phosphorus-limited culture had a completely different metabolic footprint than the phosphorus-surplus culture, with hundreds of potential compounds characteristic for the respective conditions. In concomitance with the secretion of the colored compound into the medium we were able to measure a significant increase of the soluble iron. This suggests that the cells are able to release either a compound which contains iron or a compound which could mobilize insoluble iron.

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503A Medium optimization and production of the wild type pesticide degrader strain Aminobacter MSH1 in a Bioreactor Nadja Schultz-Jensen*1, Berith Elkær Knudsen2, Zuzanna Frkova3, Jens Aamand2, Jette Thykaer4, Sebastian Reinhold Sørensen2 1National Geological Survey of Greenland and Denmark (GEUS), Denmark, 2National geological Survey of Greenland and Denmark (GEUS), Denmark, 3Department of Environment Science, Denmark 4Department of Systems Biology Technical University of Denmark Denmark

The potential of the wild type pesticide degrading strain Aminobacter MSH1 for growth in an aerobic fermentation process was evaluated. The investigation included the development of an optimized growth medium (MgSO4, CaCl2, FeSO4, NH4Cl, trace elements, buffer components Na2HPO4, KH2PO4) suitable for shake flask and bioreactor cultivations of Aminobacter MSH1. The composition of the optimized mineral salt medium was compared to an elementary analysis of Aminobacter MSH1. Interestingly the strain contained more iron (0.09%) compared to the average composition of bacteria (0.01%).

Growth optima of Aminobacter MSH1 in the optimized medium were found at pH 7 and 25 °C. The influence of the medium ionic strength as well as the consumption of different C-sources (glycerol, glucose, fructose, succinate, acetic acid) on growth was also investigated.

A stimulation of BAM (2,6-dichlorobenzamide) mineralization in the optimized mineral salt medium was also observed. In the optimized medium 50 % mineralisation of BAM (1 mg/L) was achieved within 30 hours while 60 hours were needed in a buffered solution (with MgSO4, CaCl2, NH4Cl). Obviously, addition of FeCl3 and trace elements had positive effects on the mineralization rate. Further investigations on the effect of the medium on the mineralization capacity of Aminobacter MSH1 are presently underway.

504A The inhibitory potential of silver-zinc nanoparticles on Aspergillus parasiticus Ebrahim Sedaghati*1, Saied Mollayi1, Fatemeh Rostami1, Nader Doraki2 1Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan, Iran, 2Rafsanjan Food Control Laboratory (RFCL), Iran

Aspergillus parasiticus is component of storage hazardous fungi that contaminates food and feeds, also produces different Aflatoxins. Aflatoxins are secondary metabolites produced by species Aspergillus flavus, A. parasiticus and few related species. In this study, we investigated the antifungal effects of Ag-Zn nanoparticles on two isolates of Aspergillus parasiticus. The Ag-Zn nanoparticles were produced by the high voltage arc discharge method. Solid and broth media of Potato dextrose (PD) and Czapek Dox (CZ) contain 500,750, 1000 and 1250 ppm of nanoparticles was applied. On the CZ agar and PDB medium all the concentrations had significantly reduction of colony diameter after 7 days. In both broth media there was no mycelial greets in all mentioned concentrations after 7 days therefore lower concentration of nanoparticles with 25, 50, 100, 200 and 400 ppm were applied. The 400 ppm concentration is inhibitory border. Spores were treated with all above concentration to assay the effects of these nanoparticles on spore germination. The 400 ppm concentration on CZ was inhibitory concentration of spore germination. The lower concentration had spore germination and higher concentrations were completely inhibitor. Our results suggest the possibility of using silver-zinc nanoparticles as an alternative to chemical compounds and medicine on A. parasiticus, and they are able to inhibit strongly the fungal growth and spore germination.

505A Key aromatic dioxygenases are involved in the catabolism of hydroxylated- benzoates and -phenylacetates in Burkholderia xenovorans LB400 Michael Seeger*, Romero Maria Jose Universidad Tecnica Federico Santa Maria, Chile

Bacteria from Burkholderiales order are commonly able to degrade a large number of aromatic compounds. Burkholderia xenovorans strain LB400 is a model polychlorobiphenyl-degrading bacterium. Genome analyses of strain LB400 have revealed a large number of central and peripheral catabolic pathways to degrade aromatic compounds. In this study, a survey on functional ring-cleavage steps during hydroxylated benzoates and phenylacetates catabolism by B. xenovorans LB400 is presented. Growth studies showed that B. xenovorans LB400 is able to mineralize 3-

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hydroxybenzoate (3-HBA), 4-hydroxybenzoate (4-HBA), 3-hydroxyphenylacetate (3-HPA) and 4-hydroxyphenylacetate (4-HPA) as sole carbon source. Related ring-cleavage pathways to HBAs and HPAs peripheral reactions were studied in strain LB400. Strain LB400 was able to degrade gentisate, protocatechuate, homogentisate and homoprotocatechuate, suggesting functional dioxygenase-encoding genes. Transcriptional analyses of genes encoding key aromatic dioxygenases were performed during growth on HBAs and HPAs. Expression of mhbD gene encoding a gentisate 1,2-dioxygenase was observed in 3-HBA-grown cells. The hmgA gene encoding a homogentisate 1,2-dioxygenase was expressed during growth on both HPAs, whereas a second hmgA gene was only expressed during growth on 3-HPA. Expression of pcaG gene encoding the protocatechuate 3,4-dioxygenase alpha subunit was observed during growth of strain LB400 on 4-HBA. The hpaD gene encoding a homoprotocatechuate 2,3-dioxygenase was expressed during growth on both HPAs. These results indicate that expression of key aromatic dioxygenases is up-regulated during growth of strain LB400 on HBAs and HPAs. Furthermore, a gentisate 1,2-dioxygenase activity was observed in cells grown on 3-HBA. A higher homogentisate dioxygenase activity was measured in 3-HPA- and 4-HPA-grown cells compared to glucose-grown cells. LB400 cells grown on 4-HPA showed a high protocatechuate 3,4-dioxygenase activity. A high homoprotocatechuate dioxygenase activity was measured during growth of strain LB400 on 3-HPA and 4-HPA. These results indicate that in strain LB400 3-HBA and 4-HBA are channelled into gentisate and protocatechuate central pathways. On the other side,3-HPA and 4-HPA separately funnel into both homogentisate and homoprotocatechuate central pathways. This study reveals the great metabolic machinery of B. xenovorans LB400, in which diverse key aromatic dioxygenases are used by this bacterium during growth on aromatic isomers.

506A Molecular insights into anaerobic biological dehalogenation - An important environmental process unraveled by combined genomic, biochemical and physiological studies Jana Seifert1, Goris Tobias2, Adrian Lorenz3, Boll Matthias4, Lechner Ute5, Nijenhuis Ivonne3, Sawers Gary5, Schubert Torsten2, Seifert Jana*3, Tarkka Mika3, von Bergen Martin3, Wubet Tesfaye3, Diekert Gabriele2 1UFZ-Helmholtz Center for Environmental Research GmbH, Germany, 2Friedrich Schiller University Jena, Germany, 3UFZ-Helmholtz Center for Environmental Research, Germany, 4University Leipzig, Germany, 5Martin-Luther-University Halle, Germany

Organohalides of natural or anthropogenic origin are found in many different habitats, sometimes in high concentrations. A lot of them are ecological harmful, often also toxic and/or carcinogenic. Dehalogenation of most of these compounds occurs, if at all, only at slow rates and mainly due to anaerobic metabolic activities of bacteria. In order to shed light on this process, the DFG research network FOR 1530 was established to investigate the biochemistry and physiology of the anaerobic degradation of organohalides. Organisms like Sulfurospirillum multivorans or Dehalococcoides spp. are able to couple dehalogenation to energy conservation via the generation of a proton gradient across the membrane. This anaerobic respiration is designated organohalide respiration. Additionally, co-metabolic processes may be responsible for dehalogenation, as in e.g. Thauera chlorobenzoica. The key enzymes of organohalide respiration are reductive dehalogenases (Rdhs), which are corrinoid-dependent, iron-sulfur cluster containing enzymes.

Several different methods are used to study these special metabolic pathways and the dehalogenases involved. Genome sequences of Sulfurospirillum and Dehalococcoides strains are available to complement the biochemical and genetic investigations such as proteomics, transcript level quantifications, isotope fractionation and various spectroscopic methods. The genome sequencing and annotation of S. multivorans and the Dehalococcoides strains BTF08 and DCMB5 have already been finished and yield insight into the genetic background of organohalide respiration including the electron-delivering enzymes (e.g. hydrogenases) and electron-transferring compounds. The Dehaloccocoides genomes revealed a similar genetic core architecture as already described for several Dehalococcoides strains. High-plasticity regions encoding 20 and 23 non-identical Rdhs in strain BTF08 and DCMB5, respectively, might be responsible for their specific substrate spectra (see poster Pöritz et al. in the session Pathways and Physiology). S. multivorans seems to encode a different machinery responsible for organohalide respiration. Adjacent to the characterized PCE-reductive dehalogenase PceA, genes for a second reductive dehalogenase have been found. Both have an associated pair of genes encoding a two-component system. A complete set of genes encoding proteins for corrinoid biosynthesis is found on the same gene cluster. Situated in between are genes coding for a putative quinol dehydrogenase, which may connect the reductive

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dehalogenases to the quinone pool in Sulfurospirillum. These napGH-like genes are not found in any other sequenced organohalide-respiring bacterium. Purification and heterologous expression studies in combination with the aforementioned methods will allow further insight into the nature of the reductive dehalogenases and the electron transport chain of these organisms.

The results contribute to our understanding of the different mechanisms of anaerobic biological dehalogenation at the molecular level. This will lead to a better understanding of the ecological impact of this fascinating microbial degradation capability and its role in the global halogen cycle. Additionally, it provides considerable information that may prove helpful in the application of this process for bioremediation of sites contaminated with halogenated compounds.

507A Organohalide respiring Firmicutes – from dedicated degraders to all-rounders Hauke Smidt*, Thomas Kruse, Farai Maphosa, Tian Ye Wageningen University, Netherlands

Organohalide-respiring bacteria (ORB) couple reductive dehalogenation of environmental pollutants at dedicated enzymes to energy conservation. Although this type of energy metabolism is widespread across several deeply branching phyla, enzymes and regulatory mechanisms seem more stringently conserved. Hence, organohalide respiration has been proposed as an evolutionarily ancient and comparatively basal type of respiration. Whereas a number of genomes are now available or in production for several isolates within the Chloroflexi (Dehalococcoides, Dehalobium) and Delta-Proteobacteria (Anaeromyxobacter, Geobacter), only limited efforts have been undertaken towards ORB belonging to the Firmicutes. In fact, only two genomes of strains of Desulfitobacterium hafniense are currently available. To fill this knowledge gap, we recently successfully initiated the elucidation of 12 genome sequences of organohalide-respiring Firmicutes within the genera Dehalobacter (3 strains) and Desulfitobacterium (9 strains), which differ in their dehalogenating activities and eco-physiological features, such as isolation source, electron donors, and alternative electron acceptors. In drastic contrast to the situation in Dehalobacter spp., which are dedicated degraders that are restricted in their energy metabolism to organohalide respiration, Desulfitobacterium spp. isolates known to date are all very versatile and can use a large variety of alternative electron donors and electron acceptors for growth. Sequencing is currently realized in the framework of a Joint Genome Institute Community Sequencing Project. State-of-the-art comparative and functional genomics approaches are used to unravel and exploit strain-level genomic plasticity and adaptation within members of organohalide-respiring Dehalobacter and Desulfitobacterium, capitalizing on this large-scale sequencing effort. Of specific interest in this respect is also the non-dehalogenating human intestinal isolate Desulfitobacterium hafniense DP-7, the genome of which has been sequenced in the framework of the Human Microbiome Project, revelling the complete absence of known reductive dehalogenase encoding genes. Furthermore, interactions of the hexachlorocyclohexane-transforming Dehalobacter sp. with Sedimentibacter sp. in an obligate syntrophic co-culture will be discussed.

508A Identification of a robust bacterial haloacid uptake system Jimmy Tsang*, Xianbin Su School of Biological Sciences, The University of Hong Kong, Hong Kong

Haloacids such as monochloroacetate were generated incidentally in large amount during disinfection of water. Many of these halogenated compounds are toxic and mutagenic. Bacteria capable of degrading these haloacids can be found naturally. These bacteria usually possess enzymes known as dehalogenase that mediated the removal of halogen from the alkanoic acid backbone. Characterization of a dehalogenase (Deh4a) from strain MBA4 of a Burkholderia caribensis has identified the presence of an associated transporter gene (deh4p) downstream of the dehalogenase gene. The expression of this haloacid operon is chloroacetate inducible. While E. coli expressing this transporter has an enhanced haloacid uptake activity, a B. caribensis mutant with a disrupted deh4p grew similarly like the wildtype. Analysis of haloacid uptake of this mutant showed that it retained 70% of wildtype activity. A Southern blot analysis has identified another genomic region that shows similarity to deh4p. A BLAST search on a draft genomic sequence has found dehp2 with 72% identity. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression of these two genes. While deh4p was induced nearly a hundred-fold in medium containing chloroacetate the induction of dehp2 was merely 7.5-fold, in the wildtype. In order to characterize these two transporters and their roles in haloacid uptake, single mutants were generated and analyzed. Plasmid

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pKNOCK-Cm containing a chloramphenicol resistant gene was used for gene disruption. Both single disruptants retained 70% of wildtype haloacid uptake activity. In deh4p‒ disruptant the induced expression of dehp2 was increased to 14-fold of wildtype uninduced level. In dehp2‒ disruptant the relative induced level of deh4p was 140. Competing solute analysis was also used to characterize the substrate specificity of the two transporters. While both permeases were able to transport haloacetates and acetate, Dehp2 also has a preference for chloropropionate and lactate. A double disruptant was also constructed from a single disruptant with pKNOCK-Km carrying a kanamycin resistant marker. The deh4p‒ dehp2‒ double mutant can still utilize chloroacetate as a growth substrate and retained 40% of induced haloacid uptake activity. A comparison between the amino acid sequences of Deh4p and Dehp2 has identified three motifs. When these motifs were used to search for similar proteins there are at least sixteen putative transporters identified in MBA4. Although chloroacetate has been confirmed to be toxic and affected the citric acid cycle it seems to be treated as an important growth substrate in B. caribensis MBA4. In order to ensure the availability and utilization of this carbon and energy source for growth a robust haloacid inducible transporter system is seemingly present.

509A Casting a net: filaments produced by Microcystis sp. in field and laboratory populations Gad Weiss*1, Moshe Harel1, Einat Daniel1, Avraham Wilenz1, Ora Hadas2, Assaf Sukenik2, Bojan Sedmak3, Elke Dittmann4, Sergei Braun1, Aaron Kaplan1 1The Hebrew University of Jerusalem, Israel, 2Israel Oceanographic and Limnological Research, Israel, 3National Institute of Biology, Slovenia, 4University of Potsdam, Germany

The reasons for the apparent dominance of the toxic cyanobacterium Microcystis sp. indicated by its massive blooms in many fresh water bodies are poorly understood. We show that in addition to a large array of secondary metabolites, some of which are toxic to eukaryotes, Microcystis sp. secretes large amounts of filamentous exopolysaccharides that form extremely long fibers several mm in length. This phenomenon was detected in our field and laboratory cultures of various Microcystis strains. In addition, we have identified and characterized three of the proteins associated with the filaments and the genes encoding them in Microcystis sp. PCC 7806 but were unable to completely delete them from its genome. Phylogenetic analysis of the most abundant one, designated IPF-469, showed its presence only in cyanobacteria. Its closest relatives were detected in Cyanothece sp. strains where the genomic organization of the IPF-469 is conserved. IPF-469 and the other two proteins identified here, bearing a haloperoxidase and an oscillin may be part of the filaments secretion pathway. We raise the possibility that the filaments help Microcystis sp. to form a dynamic biofilm affecting their microenvironment and migration in the water column, thereby helping them to adjust to their physiological needs.

510A Mercury methylation by sulfate reducers: assessment of a high-throughput method for isolation and physiological characterisation Colin Yannick*, Marisol Goñi-Urriza, Jérome Gury, Pierre Caumette, Rémy Guyoneaud Université de Pau et des Pays de l'Adour, France

Methylmercury is a well-known human neurotoxin that accumulates along the aquatic food webs. Its production from divalent mercury is tightly correlated with dissimilatory sulfate reduction in coastal anoxic sediments. To date, due to technical limitations that relied on cultivation and chemical analysis, only few microorganisms were tested for their mercury methylation capacities, and most of them are related to the Desulfovibrionaceae. Based on former studies, half of the sulfate reducers tested methylate mercury. Methylation potentials seem not to be species dependent, but rather strain dependent. Thus, the risk assessment of methylmercury production in aquatic ecosystems cannot be simply achieved by investigating the microbial diversity. In vitro cultivation needs to be improved in order to: 1- access a more complete vision of this functional group in sediments, 2- further determine their metabolic capacities, 3-evaluate the phylogenetic distribution and pathways of mercury methylation activity.

Therefore a high-throughput dilution-to-extinction procedure was applied in 384-wells microplates for sulfate reducers isolation from the Adour estuary sediments (French Atlantic Coast). Reduction of culture volumes down to 100 mL and a rapid handling of cultures led to the growth of 177 sulfate reducing strains. Based on a 98% cutoff for 16S rRNA sequences, strains clustered into 29 OTUs and 15 phylotypes constituted new cultivated taxa. The extended cultural dadaset provided a

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complementary view to diversity assessed by classical cloning and sequencing dsrAB genes (158 clones analyzed). In addition, the miniaturized cultivation ensured the metabolic characterisation of numerous isolates afilliating the Desulfobulbaceae family.

Besides, the cultivation approach was combined to a recombinant luminescent biosensor sensitive to methylmercury. This tool constitutes an alternative to the sophisticated but time-consumming chemical analytical setup tradionnally used to determine microbial mercury methylation. Results demonstrated that the sensor can be applied as a rapid test to further examine the phylogenetic distribution of methylating bacteria. Beside, high-throughput cultivation and characterisation permit together to increase our knowledge on mercury methylation pathways. Indeed, mechanisms of mercury methylation under different growth conditions (that is sulfate reduction, pyruvate fermentation or furmarate respiration) have been explored using Desulfovibrio sp. BerOc1 as a model strain. On the long view, the analysis of sulfate reducers and others microbial groups should provide us a better view of mercury speciation occurring in nature in order to evaluate risks linked to methylmercury in coastal environments.

511A Physiological and genomic characterization of a newly isolated Geobacter sp. strain AY that detoxify 1,2-dichloroethane to ethylene Naoko Yoshida*1, Kiyotoshi Asahi2, Arata Katayama3 1Toyohashi University of Technology, Japan, 2Environmental Science Institute of Nagoya city, Japan, 3EcoTopia Institute, Nagoya University, Japan

1,2-dichloroethane (1,2-DCA) is one of the most abundant C2-contaminants in aquifer environments such as ground water and river sediment worldwide, which has toxic effects on nervous system of human body. In this study, a bacterial strain capable of the dichloroelimination of 1,2-DCA to ethylene was isolated by deep-agar cultivation from a river sediment that contaminated with chlorinated ethanes and benzene. The strain named as AY had 99.7% of the highest similarity of 16S rRNA gene sequence with Geobacter lovleyi. Geobacter sp. AY utilized hydrogen, acetate, and pyruvate as electron donor for the dichloroelimination of 1,2-DCA, and Fe(III), fumarate, and 1,2-DCA as electron acceptors. Strain AY did not dechlorinate other chlorinated compounds such as 1,1,2-trichloroethane, tetrachloroethene, trichloroethylene, and 1,2-cis-dichloroethylene. The draft genome sequencing of strain AY obtained only one contig containing a putative dehalogenase-gene (rdhA) within total 478 contig (209,837 bp), suggesting the dehalogenase involved in the dichloroelimination of 1,2-DCA. The contig also contained genes cording a membrane anchor protein rdhB, a transcription factor rdhC, a trigger protein rdhT, and all genes had the highest similarities with those of Dehalobacter sp. strain WL, a 1,2-DCA dechlorinating bacteria of phylum Firmicutes. The similarities with those genes of WL were quite higher (for example 96.9% of rdhA similarity in amino acid sequence) than G. lovleyi strain SZ (for example 38.3 % of rdhA similarity). The results showed that these dehalogenase-relative genes are horizontally transferred to bacteria over different phyla.

512A Isolation of an L-amino acid oxidase-producing marine bacteria and analysis of its physiological and biochemical properties Zhiliang Yu*, Hua Qiao, Ning Zhou College of Biological and Environmental Engineering, Zhejiang University of Technology, China

L-amino acid oxidase (LAAO; EC 1.4.3.2) is usually a flavoproteinand is able to catalyze the stereospecific oxidative deamination of L-amino acids to a-keto acids, NH4

+ and H2O2. The studies have indicated that LAAOs have broad bioactivities and may act as defence or attack weapons via the H2O2 or as ideal molecular mechanisms for the acquisition of nitrogen from diverse amino acid sources. This enzyme is widely distributed in nature, but little is reported about LAAOs from marine microorganisms. In this study, a total of 157 pure isolates, including 32 actinomycetes, 51 fungus and 74 bacteria, were obtained from the intertidal zone of Dinghai sea area located in Zhoushan, Zhejiang province, China and morphologically identified. According to the results of measurement of a-keto acids, NH4

+ and H2O2 existing in fermentation supernatant with L-amino acids as substrates, only one isolate (B3) showed the capability of producing LAAO. Further studies suggested that B3 can catalyze ten out of the fifteen tested L-amino acids as substrates, indicating B3 has a broad substrate spectrum. Physiological and biochemical analysis together with molecular identification by constructing phylogenetic tree based on 16S rDNA indicated that B3 has high identity and homology with

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Pseudoalteromonas spp. with the highest sequence identity of 98.5% to Pseudoalteromonas viridis. Therefore B3 isolate was identified as Pseudoalteromonas sp. B3. The molecular cloning of this LAAO showed that the open reading frame is 1608 bp in length encoding the protein of 535 amino acid residues, including a flavin protein conservative region R-x-G-G. The deduced molecular mass of LAAO is about 60kDa, and theoretical isoelectric point is around 6. The amino acid composition, cleavage sites, secondary structure and tertiary structure of LAAO were analyzed and predicted with bioinformatics software to show that it lacks signal peptide and contains 224 alpha helixes, 72 extended strands and 239 bend regions. This study lays the foundation for further investigation on enzymatic properties, structure, biological function and application of LAAOs from marine microorganisms.

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