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Metabolomic Impact of the Human Intestinal Microbiota

Marcobal, A., Kashyap, P., Nelson T., Spormann, A.M., Sonnenburg, J. Stanford University

Small molecule metabolites produced by the microbiota are important mediators of microbe-

microbe and microbe-host interactions. New technology in mass-spectrometry offers the ability

to perform extensive characterization of the ‘universe’ of small molecules produced by the

microbiota or via microbiota-host interaction. We are applying these metabolomic approaches to

understand how the gut microbiota influences the metabolite profile of urine or feces. We have

used a humanized mouse (HM) model in which germ free (GF) mice are colonized with a

complete human intestinal microbiota, in order to search for biomarkers of microbial activity in

host urine and fecal samples. Urine and fecal samples from GF and HM mice (n=3 per group)

were subjected to analysis, using ultra performance liquid chromatography (UPLC) coupled with

high-resolution mass spectrometry (MS). Metabolomic data was analyzed and multivariate

statistical analysis was applied to determine group separation. We found a good separation

between urine and fecal metabolomes from GF mice and HM mice. In addition, we searched for

significantly different features between the groups. We assigned empirical formulas to a subset

of significantly different features using the METLIN database, and biomarker compounds of

microbial presence in the gut could be identified. For instance, the presence of microbiota leads

to higher levels of indoxyl sulphate or indoxyl glucuronide in urine in the HM group when

compared to urine from GF group. Both molecules are the products of hepatic transformation of

the metabolite indole, which is generated by bacterial transformation of tryptophan. Current

efforts are focused on expanding the identification of features related to the presence and

functionality of the gut microbial community.

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Characterization of Plant Growth Promoting Rhizobacteria Elisabeth Ferris*, Jennifer Okonta*, Michael Onofre*, Jessica Duong, Michael Pina, and Michelle R. Lum Department of Biology, Loyola Marymount University, Los Angeles, CA 90045 Plant Growth Promoting Rhizobacteria (PGPR) are found on the surface of plant roots, endophytically within plants, and some engage in a nitrogen-fixing symbioses inside root nodules of legumes. Our lab is interested in characterizing the types of PGPRs present in the environment and how they impact plant growth. Specifically, we are looking at the microbial community associated with dune lupine. We are also using genetic approaches to determine the mechanisms important for microbial association with the host plant, specifically that which occurs with species of Burkholderia. Lupinus chamissonis (dune lupine) is a nodulating plant of the legume family found natively in sand dunes along the California coast. In the Ballona Wetlands and El Segundo sand dunes near Loyola Marymount University, there is an ongoing effort to understand the effects of urbanization on the environment. The purpose of our study is to identify and characterize dune lupine’s microbial community and to analyze the effects of heavy metals such as zinc on the plant and its associated bacteria. As such, dune lupine nodules, which house beneficial bacteria that aid the plant in obtaining nitrogen, were isolated, surface sterilized, crushed, and plated on selective media to culture the bacterial community inside. Sequencing of the 16S ribosomal DNA identified strains of Bradyrhizobium sp., Rhizobium lusitanum, and Variovorax sp., all known to positively impact plant growth. Dune lupine plants were inoculated with the isolated bacteria, and it was found that each Bradyrhizobium and R. lusitanum bacterial strains formed nodules and Variovorax increased germination. Zinc assays showed that some of the microbial community is tolerant of at least 100 µM of zinc, although dune lupine can tolerate as much as 250 µM of zinc. Burkholderia unamae is a nitrogen-fixing species of bacteria found in endophytic relationships with crops such as maize, sugarcane, and tomato. Based on previous research, we hypothesized that motility in B. unamae will play a critical role in the bacterium’s ability to interact with plants. We generated a pool of transposon-tagged mutants and screened them for defects in motility on 0.3% agar. One of the mutants, MO384, was chosen for further characterization and was confirmed to be non-motile. It also has an alteration in cell morphology and appears to produce excess exopolysaccharide. Molecular methods identified the mutation to be in the gene that encodes FlhC, a transcriptional activator that has been identified in other bacteria in be involved in flagellar regulation. We are currently constructing a vector to express the wild type copy of flhC in the mutant in order to complement it.

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STRUCTURE-FUNCTION ANAYLSIS OF NITROBENZENE 1,2-DIOXYGENASE Kristina M. Mahan*, Juan V. Parales, and Rebecca E. Parales Department of Microbiology, University of California, Davis Nitroaromatic compounds are toxic, synthetic chemicals commonly used in the production of pesticides, dyes, plastics, and explosives. These synthetic compounds are resistant to biological and chemical degradation, and therefore they persist in the environment for long periods of time. Only a few microorganisms have been able to adapt and evolve new metabolic pathways that utilize these man-made compounds as their sole carbon, nitrogen, and energy sources. One such microbe is Comamonas sp. JS765, a strain that is capable of completely mineralizing the toxic nitroaromatic compound nitrobenzene to carbon dioxide and nitrite. We are interested in characterizing nitrobenzene 1,2-dioxygenase (NBDO), the initial enzyme in the nitrobenzene degradation pathway. NBDO is a Rieske- type enzyme system that requires specific electron transfer proteins (reductase and ferredoxin) to transfer electrons from NADH to the catalytic oxygenase component. Little is known about how the soluble protein components of Rieske dioxygenases interact in order to catalyze these unique reactions. NBDO is a model system for structure function analysis of Rieske dioxygenases because all components have been purified and characterized and the crystal structure of the catalytic component has been determined. Structures are also available for closely related ferredoxin and reductase components Using structural data we identified residues on the surfaces of the oxygenase and ferredoxin that may be involved in protein-protein interactions between the two components. Site-directed mutagenesis was used to make amino acid substitutions in the oxygenase and ferredoxin components at sites near the Rieske clusters that we predict would interfere with electron transfer. Using whole cell biotransformation assays, we found that amino acid substitutions at position 98 (changing Val 98 to Asp, Glu, or Phe) of the oxygenase and amino acid substitutions on the acidic region of the ferredoxin (changing Asp 53, Glu 62, Leu 66, or Pro 81 to Ala or Pro 65 to Lys) resulted in severely reduced enzymatic activity. Isothermal titration calorimetry is currently being evaluated to measure the difference in protein binding interactions between the wild-type and mutant forms of these proteins. These studies identify residues on the surface of the oxygenase and ferredoxin that are involved in protein-protein interactions and electron transfer.

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A single-cell genome of Thiovulum sp

Ian P.G. Marshall, Paul C. Blainey, Stephen R. Quake, Alfred M. Spormann

Stanford University Abstract: Thiovulum is a highly-motile colorless sulfur bacterium first identified in 1913, but never isolated. We used a microfluidic device coupled to a laser trap to isolate single cells from a microbial mat veil and amplify their DNA using multiple displacement amplification (MDA). 454 sequencing of Thiovulum DNA from four single cells yielded a genome of approximately2 megabases that, while divided across 222 contigs, is relatively complete in terms of its gene content. Genes encoding for enzymes involved in the reverse TCA cycle, dissimilatory nitrate reduction to ammonia (DNRA), various genes associated with sulfur and sulfide metabolism were found. In addition, numerous anabolic pathways were also identified. Genes encoding 12 different methyl-accepting chemotaxis proteins were found along with several other genes associated with chemotaxis and motility. Many phage-associated, transposase, and regulatory genes not found in the genomes of Thiovulum’s closest sequenced relatives were also identified. The Thiovulum single-cell genome is a portrait of a colorless sulfur bacterium with a metabolism similar to other sulfide-oxidizing epsilon-Proteobacteria, but with several intriguing genotypic differences.

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Clinical and environmental isolates of P. aeruginosa differentially express two

extracellular polysaccharides that provide functional redundancy during biofilm growth.

Kelly M. Colvin, Catherine Tart, and Matthew R. Parsek.

Department of Microbiology University of Washington

1959 NE Pacific St Box number 357242 Seattle, WA 98195

Extracellular polysaccharides comprise a key component of the biofilm matrix. Many organisms that are adept biofilm producers including Escherichia coli, Vibrio spp, Salmonella spp, Burkholderia spp. and Pseudomonas aeruginosa maintain the genetic material necessary to synthesize multiple types of polysaccharides. In many cases, the different polysaccharides are niche-dependent and allow the organism to thrive in a variety of environments. P. aeruginosa produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Pel and Psl. In mucoid strains, alginate is the predominant polysaccharide of the biofilm matrix. Non-mucoid strains appear to utilize primarily the Pel and Psl polysaccharides for biofilm formation. In this study, we evaluated a range of clinical and environmental P. aeruginosa isolates for their dependence on the Pel and Psl polysaccharides for biofilm development. Mutational analysis demonstrates that Psl plays an important role in bacterial attachment. However, there was significant strain-to-strain variability in the contribution of Pel and Psl to mature biofilm structure as demonstrated by microscopy and microtiter assays. PelC protein and Psl polysaccharide expression levels were measured and found to correlate with biofilm phenotypes. This analysis has led us to propose four classes of strains based upon Pel and Psl expression profiles and functional importance during biofilm growth. Our data suggest that both Pel and Psl can act as structural scaffolds for biofilm formation. To test the hypothesis if Pel and Psl are functionally redundant, PAO1, the common laboratory strain that primarily utilizes Psl in the matrix, was mutated for Psl production. We demonstrated that after extended cultivation, biofilms harvested from psl mutants up-regulate PelC expression 3-fold compared to wild-type biofilms. Evolved isolates from the psl mutant had a higher proportion of strains with elevated c-di-GMP levels. C-di-GMP is a second messenger that regulates biofilm development and transcriptionally and allosterically regulates Pel production. Interestingly, psl mutants from our clinical and environmental isolates also selected for elevated PelC expression during biofilm cultivation compared to the corresponding wild-type, suggesting a universal compensation mechanism. These evolution experiments indicate that P. aeruginosa may have an array of tools that can perform similar enough function to counteract a defect in a primary one. This feature is one example of the advantage of functional redundancy and begins to illuminate P. aeruginosa’s biofilm versatility.

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Microbial ecology of anoxygenic arsenophototrophs in alkaline, hypersaline and thermal environments

Alison Conrad and Chad W. Saltikov

Department of Microbiology and Toxicology, University of California, Santa Cruz

Anoxygenic phototrophy by bacteria is only known to use a handful of substrates, namely reduced sulfur compounds, nitrite, ferrous iron and just recently, arsenite. Arsenophototrophy was discovered in an anaerobic bacterium called Ectothiorhodospira sp. str. PHS-1 isolated from arsenic-rich hot spring microbial mats on Paoha Island, Mono Lake, a hypersaline alkaline lake in California. This phototroph appears to use a “novel” clade of arsenite oxidase, ArxA, distinct from that of the relatively well characterized AoxB. However ArxA has only been genetically confirmed in a non-phototrophic arsenite oxidizer called Alkalilimnicola ehrlichii str. MLHE-1, also isolated from Mono Lake, and PHS-1 has not proven amenable to genetic manipulations. My work has two aims. First, to study the microbial ecology of anoxygenic arsenophototrophs in arsenic-rich environments other than Mono Lake and secondly, to isolate an arsenophototroph which can serve as a genetic model for photosynthesis-linked arsenic oxidation. Water, sediment, microbial mat and tufa collected from Big Soda Lake (Churchill County, NV), Mono Lake and various hot springs in the Mammoth Lakes area were used for enrichment culturing and functional gene analyses. Currently, the results show that arxA-like genes appear to be widely distributed in Big Soda Lake. Moreover, several isolates obtained from Big Soda Lake contain arxA-like genes. This work supports the hypothesis that ArxA is not limited to the Mono Lake area. It is also a necessary stepping stone in actualizing a genetic model for photosynthesis linked arsenite oxidation.

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EFFECTS OF CULTURE CONDITIONS ON TOXIN PRODUCTION BY CLOSTRIDIUM DIFFICILE Xiang He Lei and Barry R. Bochner*. Biolog, Inc., Hayward, CA, USA Among infectious agents, Clostridium difficile is a major cause of morbidity and mortality in US hospitals. It is a spore-forming anaerobic bacterium capable of producing two toxins (A and B). The toxins are important determinants of C. difficile virulence, but very little is known about how toxin production is regulated by culture conditions. In this study we developed simple, reliable, and efficient cytotoxicity assays for C. difficile toxin that could be performed in standard microtiter plates. Then we systematically examined the effects of culture conditions on toxin production after culturing cells for 3 days in 100 μl cultures in Phenotype MicroArray (PM) panels. Strong effects on C. difficile toxin production were seen with certain carbon and nitrogen sources. Several amino acid carbon sources showed strong inducing effects. Among these, D-threonine gave highest toxin production followed by L-serine, L-alanine, and D-serine. Acetyl amino sugars or amino sugars as carbon or nitrogen sources (e.g., N-acetyl-D-glucosamine) showed a strong or above average stimulation effect. Fumaric acid and bromo-succinic acid gave highest toxin production among the carboxylic acid carbon sources. Among nitrogen sources tested, peptides had stronger stimulatory effects on toxin production than single amino acids. Arginine peptides gave among the highest levels of toxin production. These results provide valuable insights into metabolic regulation of toxin production by C. difficile. Furthermore, this same approach can be used with any toxin-producing microorganism to efficiently test nearly 2,000 culture conditions, some of which may induce toxin production. Therefore it can be used, not only as a way to study toxin regulation, but also as a way to discover novel toxins and other secondary metabolites produced by microorganisms.

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Packaging Metabolism: Why put enzymes in cages?

Douglas L. Huseby and John R. Roth

Department of Microbiology, University of California, Davis Ethanolamine metabolism is one of a small set of conserved functions that differentiates Salmonella species from other closely related Gammaproteobacteria (including E. coli). Other functions in this set are degradation of 1,2-propanediol, synthesis of B12, and use of the sulfur compound tetrathionate as an electron acceptor. Salmonella’s B12-dependent use of ethanolamine and 1,2-propanediol is unusual in that the necessary metabolic enzymes are contained in a proteinaceous shell, called a microcompartment. Homologous compartments, called carboxysomes contain Rubisco in cyanobacteria and function to concentrate CO2. The function of microcompartments in ethanolamine metabolism remains uncertain, but several roles are being entertained – any or all of or which may contribute. It is generally agreed that the protein cage of a microcompartment blocks passage of small, uncharged molecules, a task for which lipid membranes are ill suited. This fits the model of CO2 concentration in cyanobacteria, and seems consistent with retention of the volatile intermediate, acetaldehyde, in Salmonella. Genetic evidence supports the idea that Salmonella microcompartments sequester the large cofactors used in ethanolamine degradation -- NAD+/NADH and CoA. The B12-dependent enzyme ethanolamine ammonia-lyase, converts ethanolamine to microcompartment-internal acetaldehyde, some of which is reduced (to excreted ethanol); and some is oxidized (to acetyl-CoA) and on to microcompartment-external Acetyl-PO4. Thus the compartment is a fermentation reactor in which private internal pools of CoA and NAD+/NADH are recycled. This allows optimization of pool sizes and couples the oxidation and reduction of NAD+/NADH. We propose as a general model that all microcompartments serve to confine and concentrate volatile substrates. They increase the efficiency of the pathway by using private internal pools of substrates and cofactors. The compartments may also maintain local concentrations of enzymes and cofactors to help cells transition between times of substrate availability.

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H2 is not an essential intermediate for hydrogenotrophic methanogens

Kyle C. Costa1,2, Thomas J. Lie1, and John A. Leigh1,2

Department of Microbiology1 and Astrobiology Program2, University of Washington,

Seattle, WA

Biological methanogenesis occurs through four two-electron reductions that reduce CO2 to CH4. Members of the hydrogenotrophic (class I) methanogens are capable of growth using H2, formate, or secondary alcohols as electron donors for methanogenesis. Regardless of substrate, growth of class I methanogens is thought to require H2 as an obligate intermediate, suggesting hydrogenase is essential. Consistent with this, there is no evidence of a class I methanogen growing in the complete absence of hydrogenase. However, using the model methanogen Methanococcus maripaludis, we have constructed such a strain that lacks the active site subunits for all seven hydrogenases (Vhc, Vhu, Frc, Fru, Hmd, Eha, and Ehb). The mutant grows with formate as the sole electron donor for methanogenesis in medium supplemented with amino acids. Growth of the mutant is slower than wild-type M. maripaludis, suggesting H2 as a preferred, but not required, intermediate. The requirement for amino acids suggests the importance of H2 for anabolic CO2 fixation in methanogenic archaea. In vitro experiments with cell extracts verified that H2 is not required for generation of methane in the presence of formate. The ability of such a mutant to exist suggests novel mechanisms to generate reduced electron carriers in a H2 independent manner. Additionally, the phenotype of the mutant suggests that alternative electron donors, such as formate, are closely integrated into the methanogenic pathway. These data have profound implications for our understanding of both the role of H2 in methanogenesis as well as the substrate flexibility of class I methanogenic archaea.

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Regulation of Nitrogen Fixation by Formation of a Multimeric Inhibitory Complex Thomas J. Lie1, Kyle C. Costa1, Jeremy A. Dodsworth1, Hongjin Zheng2, Tamir Gonen2, and John A. Leigh1 Departments of Microbiology1 and Biochemistry2, University of Washington, Seattle, WA Any expensive and conditional metabolic process is bound to be rigorously regulated, and nitrogen fixation is no exception. Only when dinitrogen is the only available nitrogen source will a diazotroph expend the eight electrons and sixteen ATPs needed to convert it to ammonia. Regulation is accomplished at two levels: transcription of the nif genes and reversible inactivation of nitrogenase. The latter process, termed switch-off, occurs widely across the diversity of diazotrophs. However, until recently the mechanism was known only in the α-Proteobacteria: covalent ADP-ribosylation of the electron-donating iron protein (FePro). We found a second, completely different mechanism of switch-off in the methanogenic Archaeon, Methanococcus maripaludis. There, a protein called NifI binds directly to the molybdenum-iron protein (MoFePro, where dinitrogen reduction takes place), and apparently blocks its interaction with FePro. NifI is related to the nearly ubiquitous (in Bacteria and Euryarchaeota) PII family of 2-oxoglutarate sensor proteins that regulate a wide variety of nitrogen assimilation functions. Accordingly, NifI responds to intracellular 2-oxoglutarate as a signal of nitrogen starvation and ATP as a signal of energy status. However, NifI differs from PII proteins phylogenetically and structurally. The two subunits of NifI, NifI1 and NifI2, constitute a subfamily separate from all other PII proteins. And while PII proteins are homotrimeric, NifI is heterohexameric. Furthermore, when 2-oxoglutarate is high and NifI detaches from MoFePro, it oligomerizes to a heterododecamer. But the complex of NifI with MoFePro must be even more extraordinary, since it has to explain two observations: First, only one molecule of NifI is required to completely inhibit a molecule of MoFePro, even though the latter is symmetrical with two binding faces for FePro and two catalytic centers. Second, the NifI-MoFePro complex is much larger than what would contain a single molecule of each protein. This led us to a model in which NifI is divalent (has two identical faces like MoFePro) and alternates with MoFePro in a multimeric complex. I will present preliminary data suggesting the model is correct, and that the complex is circular. The mechanism is evidently widespread as nifI genes are present in a variety of Bacteria and Euryarchaeota.

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The Pivotal Twin-Histidine Element of the Escherichia coli Ammonium Channel AmtB

Functions as a Substrate Selectivity Filter

Jason Hall1 and Dalai Yan2

1Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0374 2Department of Microbiology and Immunology, Indiana University School of Medicine,

Indianapolis, IN 46202-5120

Ammonium functions as both a primary nutrient and waste product and, thus, its transport across

biological membranes is of fundamental importance. Because the uncharged form, NH3, readily

traverses phospholipid bilayers by simple diffusion the role of protein-catalyzed transport of the

protonated species, NH4+, is unusually interesting. The Amt family of channels mediates the

transport of NH4+ and is required for microbial growth when diffusion of NH3 becomes limiting

for nitrogen uptake. Whereas all other characterized channels facilitate downhill substrate

movement, Amt proteins are active channels – hybrids between passive channels and active

transporters – and concentrate NH4+ against a gradient. Amt family members function as

homotrimers, with each monomeric unit carrying a pore for substrate conduction. Each pore is

lined entirely with hydrophobic residues, save for a pair of conserved hydrogen-bonded

histidines postulated to play a critical role in mediating NH4+ transport. We examined the impact

that changes to this histidine pairing had on the function of one of the best-characterized

members of the Amt family, the AmtB protein of Escherichia coli. Our initial analysis indicated

that AmtB can accommodate, by either direct substitution or suppressor generation, acidic

residues at one or both positions of the twin-histidine site while retaining good-to-excellent

transport activity. Subsequent work shows that a number of mutant AmtB proteins carrying such

alterations leak K+ ions and that this leakage is energetically costly. These findings lead us to

conclude that although the twin-histidine element is not required to conduct NH4+, it serves as a

filter to prevent AmtB-mediated K+ transport.

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Anaerobic nitrate respiration provides a growth advantage for Enterobacteriaceae during Non-infectious inflammatory diarrhea Sebastian E. Winter1, Maria G. Winter1, Mariana N. Xavier1, Parameth Thiennimitr1, Valley Stewart2, Renée M. Tsolis1 and Andreas J. Bäumler1 1 Department of Medical Microbiology and Immunology, UC Davis, Davis CA. 2 Department of Microbiology, UC Davis, Davis CA. During flare-ups of inflammatory diarrhea in inflammatory bowel disease (IBD) patients, the overall composition of the intestinal microbiota changes significantly. While strictly anaerobic members of two major phyla Bacteroidetes and Firmicutes are depleted, members of the Enterobacteriaceae are enriched during disease episodes. The mechanisms driving these changes in the microbial community structure remain uncharacterized. Here we present evidence that highly oxidized compounds generated as a byproduct of the inflammatory host response can serve as alternative electron acceptors to support growth of Enterobacteriaceae by anaerobic respiration. E. coli was used as an indicator for growth of Enterobacteriaceae in a murine model of non-infectious colitis. Nitrate respiration proficient E. coli strains efficiently outcompeted isogenic, nitrate respiration deficient mutants in the inflamed gut. This growth advantage was abrogated in Nos2-deficient mice, suggesting that the generation of nitric oxides by inducible nitric oxide synthase (iNOS) is required for generating nitrate and supporting growth of E. coli by nitrate respiration in the inflamed intestine. We conclude that during non-infectious, inflammatory diarrhea, highly oxidized compounds are produced during inflammation and confer a substantial growth advantage for Enterobacteriaceae over other members of the microbiota that solely rely on fermentation to obtain energy for growth.

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BipA is a translation factor that is required for ribosome assembly in E. coli.

Lolita  Piersimoni1,2,  Ryszard  Zielke1,  Mengxi  Jiang1,  Claudio  Gualerzi2,  Janine  Maddock1  1Department  of  Molecular,  Cellular  and  Developmental  Biology.    University  of  Michigan,  USA.    2Department  of  Biology  MCA,  University  of  Camerino,  Italy.   BipA is a ribosome-associated GTPase most closely related to the elongation factor EF-G. The function of BipA, however, is currently unknown. We show that in vitro, BipA stimulates translation on a model mRNA, indicating that BipA plays a positive role in translation. Although bipA is not an essential gene, ∆bipA mutants are quite cold sensitive. One possibility is that BipA is required for the optimal translation of one or more mRNAs at cold, but not warm, temperatures. While searching for these BipA-dependent mRNAs, we found that the ∆bipA mutant had a severe ribosome assembly defect. At 18°C, we find that both immature 30S and 50S particles accumulate. The phenotype is striking at low cell densities but, at higher cell densities, the ribosome profiles are normal, suggesting that BipA is only required during active growth in the cold. For the large subunit, a very early pre-50S particle, the 32S, accumulates. We have been characterizing the nature of the 32S particle using both conventional immunoblotting as well as quantitative proteomic approaches. Curiously, our data are somewhat contradictory to the well-established in vitro assembly pathway. For example, the 32S particle lacks L20 but contains L24, both early binding proteins. Thus, we predict that ribosome assembly in the bipA mutant differs from that previously seen in vitro.

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The underlying mechanisms of social gliding motility by Clostridium perfringens and its role in invasive disease Andrea Hartman, Brittany Gianetti and Stephen Melville Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061 We have recently discovered that Clostridium perfringens, and likely all other Clostridia, can move across surfaces using a unique type of social gliding motility. Type IV pili (TFP) have been shown to be important for this group behavior. Using a combination of immunofluorescence and GFP-protein fusion experiments, we found that the pili and pilus assembly proteins are located at the poles of the cells. In-frame markerless deletions of each gene encoding a putative pilin protein were made. Each of these mutants still made the end-to-end connections characteristic of C. perfringens gliding motility but did show reduced adherence to murine muscles cells. We also constructed mutations in genes encoding sortase-dependent fimbrae in C. perfringens and found that these were defective in both social gliding motility and adherence to mouse muscle cells. A comprehensive model will be presented illustrating how these features combine to give C. perfringens the ability to move rapidly and destructively through animal tissues in a gas gangrene infection.

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Non-coding RNAs, newly identified players in regulating the N-metabolism of Methanosarcina mazei Gö1 Jäger D1, Ehlers C1, Weidenbach K1, Backofen R2, Sharma CM3, Vogel J3, Schmitz-Streit R1

1 Department of Gerneral Micobiology, Christian-Albrechts-University Kiel, Germany 2 Bioinformatics Group, Department of Computer Science, Albert-Ludwigs-University Freiburg, Germany 3 Research Center for Infectious Diseases (ZINF), University Würzburg, Germany

Small non-coding RNAs (sRNAs) have been identified in all three domains of life. In Eukarya

and Bacteria functions have been assigned for many sRNAs; in contrast still little is known on

regulatory roles and the regulatory mechanisms of sRNAs in the domain of Archaea. The

archaeal model organism Methanosarcina mazei strain Gö1 is a representative methylotrophic

archaeon of significant ecological importance due to its role in biogenic methane production in

various anaerobic habitats on Earth and is able to fix molecular nitrogen (N2). We recently

performed a genome-wide RNA-seq approach, resulting in the discovery of 248 sRNAs in M.

mazei expressed from intergenic regions, 18 of which were demonstrated to be differentially

transcribed in response to nitrogen availability [1]. Here we present the characterization of

sRNA154, which is exclusively expressed under nitrogen starvation, using biochemical and

genetic approaches. The potential regulatory role of sRNA154 in the N-regulon potentially

affecting transcriptional regulators will be discussed.

References 1. Jäger D , Sharma CM , Thomsen J, Ehlers C, Vogel J, Schmitz RA (2009) Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability. PNAS. 106(51):21878-21882

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Genetic and Biochemical Investigations of SSV1_VP2, a DNA Binding Protein from a Hyperthermophilic Archaeal Virus. Eric Iverson and Ken Stedman

Biology Department and Center for Life in Extreme Environments, Portland State University, Portland, Oregon, USA.

Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most wide-spread and best-studied are the lemon-shaped fuselloviruses. The spindle-morphology is unique to Archaea but widespread therein. The best-studied fusellovirus is SSV1 from Beppu Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Inspired by Sydney Kustu, we have undertaken a genetic and biochemical study of the genes encoded by this virus. The VP2 protein was reported to be a structural and DNA-binding protein in the late 1980s. It is very rich in basic amino acids. Interestingly the VP2 gene is not well-conserved in other Fuselloviruses. We have disrupted the VP2 gene in SSV1 and the resulting virus appears to function normally. In parallel we have expressed the VP2 protein recombinantly and have verified its DNA binding. It appears to bind DNA with extremely high affinity. Structural characterization of the recombinant protein is underway.

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A  Bacillus  simplex  strain  isolated  by  undergraduate  students  at  UCLA  promotes  plant  growth  by  procuring  soil  nutrients  and  may  also  serve  as  a  biological  control  agent    Irma  Ortiz1,  Allison  R.  Schwartz1,  Erin  R.  Sanders3,  Roky  Coria  4,  Andrew  C.  Diener1,  Darleen  A.  DeMason4  and  Ann  M.  Hirsch1,2  1Dept.  of  Molecular,  Cell,  and  Developmental  Biology,  2Molecular  Biology  Institute,  3Dept.  of  Microbiology,   Immunology,   and   Molecular   Genetics,   4Dept.   of   Ecology   and   Evolutionary  Biology  University  of  California,  Los  Angeles,  90095-­‐1606  and  5Dept.  of  Botany  and  Plant  Sciences,  University  of  California,  Riverside,  CA  92521,  U.S.A.  The  plant  microbiome  or   the   rhizosphere   consists  of  both  bacteria  and   fungi   that   exhibit  many   beneficial   effects   on   plant   growth.     The   Plant-­‐Growth   Promoting   Bacteria   (PGPB)  offer  an  important  solution  to  the  negative  consequences  of  adding  chemical  fertilizers  and  pesticides  by  enhancing  plant  yield,  producing  phytohormones  such  as  auxin  or  interfering  with   ethylene   synthesis,   controlling   plant   pathogens,   and   obtaining   critical   nutrients  including   iron,   nitrogen,   and   phosphorous.     In   this   study,   we   analyzed   the   mechanisms  whereby   a   Bacillus   strain,   isolated   from   the   Mildred   E.   Mathias   Botanical   Garden   and  investigated  by  two  undergraduate  courses,  could  mediate  a  positive  growth  response  on  the  legume  Pisum  sativum.    From  various  assays,  we  determined  that  the  improvements  in  plant   growth   based   on   dry   weight   accumulation   are   likely   to   be   the   result   of   several  bacterial   factors   including   IAA   synthesis,   siderophore   production,   and   phosphate  solubilization.    We visualized the plant’s reaction to inoculation by utilizing P. sativum lines expressing the auxin-responsive DR5::GUS promoter construct. B. simplex and Rhizobium leguminosarum bv. viciae were inoculated either singly or together. Because   PGPB   are   also  known   to   protect  plants   from  pathogenic   fungi   and   bacteria,   a   co-­‐culture   assay  was   also  established  for  these  organisms.    Culture plates were spotted with a number of different fungal pathogens, and then co-inoculated with B. simplex or B. subtilis; the latter is a well-known biocontrol agent. Our results so far indicate that the newly isolated B. simplex strain is less active as a biocontrol agent than B. subtilis.  We   acknowledge   the   financial   support   of   the   Shanbrom   Family   Foundation   and   the   UCLA   Office   of  Instructional  Development   for  courses  MIMG121A  and  MCDB150L.     Irma  Ortiz  was  supported  by  NIH  grant  GM55052  to  Dr.  Richard  L.  Weiss  (UCLA).    Other  undergraduates  who  participated  in  this  research  are  Rudy  Benitez,   Nigar   Yusifova,   Christine   Kim,   Ethan   Mathews,   Mary   Motamedinia,   Archie   McCoy,   Han   Soul   Kim,  Kayoko  Hanamoto,  Walter  Kim,  Judy  Wong,  Brittany  Yee,  and  Faith  Oh.    

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  Archaeal Surface Layer Proteins in the Methanosarcinaceae Robert P. Gunsalus, D. Francoleon, J. Loo, R. Loo, L. Rohlin, U.M. Kim, and M. Arbing. Department of Microbiology, Immunology, and Molecular Genetics, and the Department of Chemistry and Biochemistry. University of California, Los Angeles, CA The cell envelopes of many archaeal species posess a proteinaceous surface or lattice termed the surface-layer (S-layer). It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form a highly organized surface-exposed array. Currently, very little is known about the properties of such surface arrays in any archaean. Surprisingly, over a hundred proteins were annotated to be S-layer or surface associated components in the Methanosarcina mazei Gö1 genome, reflecting limitations of current bioinformatics predictions. To experimentally address what proteins are present, we devised an in vivo biotinylation technique to affinity tag all surface-exposed proteins that overcame challenges in working with these fragile microorganisms. The Methanosarcina species were adapted to growth under N2 fixing conditions to minimize the level of free amines that would interfere with the NHS-label acylation chemistry used. A 3-phase separation procedure was then employed to isolate the intact labeled cells from any lysed-cell derived proteins. The Streptavidin affinity enrichment was followed by stringent wash to remove non-specifically bound proteins, and LC-MS-MS methods were employed to identify the labeled surface proteins. In M. mazei Gö1 the major surface layer protein was identified to be the MM1976 gene product. It was shown to exist in multiple glycosylated forms by using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. We also explored the species diversity in other Methanosarcina strains: single S-layer proteins were identified in Methanosarcina barkeri strain fusaro and in Methaosarcina acetivorans C2A using related proteomic methods. Again, SDS-PAGE revealed multiple isoforms with apparent sizes ranging from 101 to134 kDa in size, indicating post-translational modifications of N-terminal protein processing and glycosylation. A family of related S-layer proteins/genes was identified in all of these Methanosarcina genomes with features related to the surface layer protein found in Methanosarcina maziei Goe1. These data reveal a conserved protein signature in the Methanosarcinaceae and with distinct protein features and implied architecture that is absent in other archaea. To address S-layer structure and function, crystallographic studies were performed: the DUF1608 domain structure of the M. acetivorans S-layer protein was determined at 2.3 A, and it reveals how the protein assembles into the cell surface lattice, allows movement of small molecules across it, and anchors the SLP to the cell lipid membrane.

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Gastric microbiota alters host immune response to Helicobacter pylori infection

Annah S. Rolig1, Cynthia Cech3, Ethan Ahler3, J. Elliot Carter2, and Karen M. Ottemann3

Departments of Molecular, Cellular, and Developmental Biology1 and Microbiology and Environmental Toxicology3, University of California, Santa Cruz, Santa Cruz, California 95064; Department of Pathology, University of South Alabama College of Medicine, Mobile Alabama 366882;

Abstract

Alterations in the microbiota composition modulate disease severity in several conditions. We

report that disease outcome from the gastric bacterial pathogen Helicobacter pylori is modulated

by pre-infection host microbiota. Utilizing a mouse model, we altered the pre-infection

microbiota using antibiotic treatment and found that microbiota alterations reduced the number

of CD4+ T helper cells responding to an H. pylori infection. Additionally, a greater percentage of

these T cells were regulatory T cells. The bacterial communities in mice with a reduced response

to H. pylori had significantly more cluster IV and XIVa Clostridia spp., bacteria known to

increase populations of regulatory T cells. Our findings suggest that microbiota composition

contributes to the variable disease outcome to H. pylori infection by promoting specific immune

cell recruitment.

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Effects of Zinc on Dune Lupine and Its Microbial Community Lisa Ferris, Jessica Duong, Jennifer Okonta, and Michael Piña Lupinus chamissonis (dune lupine) is a known nodulating species of the legume plant family found natively along the California coast. In the Ballona wetlands and El Segundo sand dunes near Loyola Marymount University, there is an ongoing effort to understand the effects of urban runoff on sand dunes, where dune lupine grows. In particular, the purpose of our study is to observe the effects of zinc on L. chamissonis and its associated bacteria. To characterize the bacteria that normally associate with dune lupine, nodules were isolated from roots of L. chamissonis, surface sterilized, crushed, and plated on selective media. Multiple bacterial strains were found and are currently being tested for growth promoting properties such as nitrogen fixation, cellulase activity, and ACC deamination. Sterile L. chamissonis plants were inoculated with the bacterial isolates and some formed nodules, confirming we had isolated dune lupine symbionts. To identify the bacterial isolates, (PCR) was performed on the 16S ribosomal DNA and the products were sequenced and compared to the database. The identification of Rhizobium lusitanum Bradyrhizobium japonicum, Methylobacterium tardum, and Arthrobacteria viscosus have been confirmed. Also, the species B. japonicum. has been found to nodulate. In addition, zinc assays are being used to test for zinc tolerance in L. chamissonis as well as the bacterial isolates. Preliminary data shows that excess zinc induces stress in bacterial strains, yet increases germination rates of L. chamissonis. This suggests that the plant may be protecting the bacteria from zinc stress. Further studies will investigate whether or not zinc is present in the nodules.

 

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Uncovering the genetic basis of 1,4-dioxane metabolism in Pseudonocardia dioxanivorans strain CB1190 Ariel Grostern (1), Christopher M. Sales (1), Wei-Qin Zhuang (1), Onur Erbilgin (2), Rebecca Parales (3), and Lisa Alvarez-Cohen (1). (1) Department of Civil and Environmental Engineering, UC Berkeley (2) Department of Plant and Microbial Biology, UC Berkeley (3) Department of Microbiology, UC Davis The cyclic ether 1,4-dioxane (dioxane) is a probable human carcinogen and emerging groundwater contaminant that is used in paper and textile processing and as a stabilizer for the solvent 1,1,1-trichloroethane. Two decades ago the first bacterium able to grow with dioxane as the sole source of carbon and energy was isolated. This actinobacterium was subsequently designated Pseudonocardia dioxanivorans strain CB1190, and in 2007 a dioxane degradation pathway was proposed based on metabolite identification. Here we present in-progress work to discover the genetic basis for 1,4-dioxane metabolism in strain CB1190. Dioxane degradation is initiated by the activity of a monooxygenase, and whole genome sequencing revealed the presence of seven multicomponent monooxygenase gene clusters. Only a single, plasmid-encoded, monooxygenase gene cluster was upregulated when strain CB1190 was grown with dioxane, as determined by microarray analysis of transcripts. When this monooxygenase gene cluster was heterologously expressed in the actinobacterium Rhodococcus jostii strain RHA1, whole cells degraded dioxane and the related cyclic ether tetrahydrofuran. Microarray analysis of dioxane-induced genes in strain CB1190 also identified how dioxane metabolites are assimilated into central metabolism – namely through the glyoxylate carboligase pathway. The activity of glyoxylate carboligase was confirmed in cell-free extracts from dioxane-grown cells. Several strain CB1190 putative aldehyde dehydrogenase and alcohol reductase genes were also induced by dioxane, and their involvement in the transformation of dioxane metabolites is currently being tested.

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Genomic Analysis of 67 Newly Sequenced Haloarchaea

Lynch EA1, Langille MGI2, Wilbanks EG1, Tritt A2, Larsen D2, Shao K2,6, Haitliner C2, Eisen JA1,2,4,5, Darling A2, and Facciotti MT1,2,3

1. Microbiology Graduate Group, University of California, Davis, 2. Genome Center, University of California, Davis, 3. Department of Biomedical Engineering, University of California, Davis, 4. Department of Evolution and Ecology, University of California, Davis, 5. Department of Medical Microbiology and Immunology, University of California, Davis, 6. Davis Senior High School, Davis, California Background: The Halobacteriacea (informally Haloarchaea) are a family of archaea thriving at high salinities and include alkaliphilic, facultatively thermophilic, thermoalkaliphilic and psychrotolerant species. The ease with which these organisms are cultured in the laboratory also makes them uniquely suited to serve as model organisms for the study of archaea. In addition, their pronounced resistance to multiple environmental stresses (radiation, metal toxicity, salinity) and unique metabolic capabilities (cellulose degradation, production of bioplastics) make haloarchaea of interest for a variety of biotechnologial and industrial applications. Methods: Sixty-seven genomes from 25 haloarchaeal genera were sequenced to an average depth of 189x coverage with a combination of Illumina GA2 and Roche 454 pyrosequencing. Errors were corrected with SGA and genomes were assembled with IDBA and SSPACE using the newly minted automated A5 assembly pipeline. A combination of RAST and IMG was used for genome annotation. Phylogeny was determined using Amphora and BUCKy. Results: At time of sequencing, only 16 haloarchaeal genomes were available from NCBI. This study therefore represents a 5-fold increase in haloarchaeal genomic data. Concurrent projects have resulted in complete genomes for an additional 18 species, making haloarchaea by far the most deeply-sequenced archaeal clade. Work on assembly of these new genomes has resulted in the construction of an automated pipeline for de novo genome assembly of Illumina data, which outperforms popular assembly tools. We report results of comparative genomic analysis, along with discovery of several interesting features of haloarchaeal biology. Conclusions: Initial analyses of a deeply-sequenced haloarchaeal clade reveals complex evolutionary patterns in protein families involved in transcriptional regulation and DNA metabolism, including intra-family and distant HGT events. We have also found evidence for genera-level life-style specialization, reflected in protein functional category ratios. Further analysis of this rich dataset will provide insights into haloarchaeal metabolism, gene regulation and genome evolution.

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Direct probing betaine microbial metabolism by ambient mass spectrometry

Marie-Pierre Forquin-Gomez1, Richard Jeannotte1, Marek A. Domin2, and Bart C. Weimer1

1Population Health and Reproduction Department, School of Veterinary Medicine, University of

California Davis, Davis, CA 95616; 2Boston College, Eugene F. Merkert Chemistry Center,

Chestnut Hill, MA 02467, USA

In the preparation of cheese and fermented products, bacteria could be exposed to

hyperosmotic conditions. Microorganisms developed different strategies to adapt to these

extreme environments by accumulating some solutes like ions, carbohydrates, amino acids, or

amino acids derivatives. The Brevibacterium genus accumulates different types of

osmoprotectant (glycine betaine, ectoine, glutamate, trehalose) whereas Lactobacillus species

accumulate some amino acids such as proline, glutamate or carnitine, an amino acid derivative,

and prefer to uptake glycine betaine from the medium. In this study, we wanted to assess the

metabolic response of these three bacteria to hypersaline stress. We performed an experiment in

which we exposed Lactobacillus acidophilus (5% NaCl), Lactobacillus plantarum (8% NaCl)

and Brevibacterium aurantiacum (11% NaCl) to high salt levels during 24 hours in the stationary

phase. The cells were then recovered and dried by centrifugal evaporation. The dried cells were

directly analyzed by a new ambient mass spectrometry technique, the Direct Analysis in Real

Time (DART) mass spectrometry. This technique allows the analysis of solid, liquid and gaseous

samples with minimal to no sample preparation. The identification is based on accurate mass of

the molecules. We used this approach to detect metabolites modulated during the osmotic stress.

Surprisingly, glycine betaine and alanine betaine levels in Brevibcaterium aurantiacum decrease

in the presence of NaCl, but ectoine concentration increased as expected. We also find two

unexpected betaine compounds in the Lactobacilli species that were regulated by osmotic stress:

alanine betaine and proline betaine. DART-MS allows to obtain a snapshot into a microbial

metabolism and could successively used to screen large sample sets. Finally, it allows us to

discover new osmoprotective compounds involved in the hyperosmotic resistance in the studied

bacteria.

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Identification of a novel polysaccharide secretion system essential for gliding motility in Nostoc punctiforme.

Douglas D. Risser

John C. Meeks

UC Davis Department of Microbiology

Nostoc punctiforme is a filamentous cyanobacterium with the ability to develop a diverse range of cell types including nitrogen-fixing heterocysts, spore-like akinetes, and the motile hormogonia. Hormogonia are short chains of morphologically distinct cells that are capable of gliding motility, a process that has yet to be fully defined. It has been postulated that gliding motility in hormogonia is driven by the secretion of polysaccharides, colloquially referred to as the “slime gun” model of gliding motility, and electron microscopy has identified a ring of putative polysaccharide secretion pores adjacent to the cell septum in motile cyanobacteria. However, to date the identity and function of these pores has not been characterized. Comparative transcriptomics and protein localization studies with a chemotaxis-like gene cluster required for motility and polysaccharide production led us to investigate a previously uncharacterized gene cluster which may encode the structural genes for these pores as well as the polysaccharide synthesis components. Deletions in three different regions of this cluster resulted in non-motile filaments that no longer secrete polysaccharide supporting the hypothesis that this cluster encodes a polysaccharide secretion system essential for gliding motility. It is possible that this system is the motor that drives gliding motility in cyanobacteria. Alternatively, while essential for motility, polysaccharide secretion may play a more accessory role, such as providing an appropriate matrix for gliding via another mechanism.

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Bacterial Perfectionists - How corrinoid producers specify the lower ligand

Terence S. Crofts, Amrita Hazra, Jennifer Tran, Erica Seth, Michi E. Taga

UC Berkeley, Department of Plant and Microbial Biology

Corrinoids such as vitamin B12 are a class of complex small molecule cofactors. While required by many animals and prokaryotes, only a subset of prokaryotes can synthesize corrinoids. These cofactors, of which more than a dozen are known, differ from each other only in the identity of their lower ligands. Interestingly, corrinoid producing organisms generally are only found to make one or two types of corrinoids in nature. We were interested in whether the specificity for particular lower ligands is due to limited lower ligand availability or if lower ligand use is actively regulated. We have identified the in vivo lower ligand specificity of five diverse corrinoid producing organisms, Sinorhizobium meliloti, Salmonella enterica, Dehalococcoides ethenogenes, Lactobacillus reuteri, and Veillonella parvula. When provided a spectrum of different lower ligands each organism only incorporated a subset of them into complete corrinoids. This indicates that lower ligand specificity is actively regulated, and not just a reflection of lower ligand availability.

The CobT family of enzymes has previously been shown to be responsible for activating lower ligands prior to being incorporated into complete corrinoids. We hypothesized that substrate specificity in the CobT enzyme could be responsible for the observed limitations in corrinoid production. To test this hypothesis we expressed the cobT genes from each organism in a cobT mutant strain of S. meliloti. Using this genetic background we again tested the ability of each lower ligand to be incorporated into a complete corrinoid. We found that in all but one case the expression of the cobT homologs was sufficient to change the specificity of S. meliloti to match the donor organism. Next, the activities of the CobT proteins were assayed in vitro. The proteins were provided each lower ligand as a substrate both one at a time and in pairs of substrates competing for enzymatic activity. The in vitro results matched what we saw when expressing cobT genes in S. meliloti and matched the original in vivo data, with one exception as before.

In summary we have shown that corrinoid producing bacteria are not significantly limited in lower ligand use by their availability. Instead, we have shown that the CobT enzyme is sufficient to explain observed lower ligand specificity. Corrinoid producers have therefore evolved to limit the types of corrinoids that they will produce. This finding is especially interesting in the context of mixed microbial communities, where we have observed a variety of known lower ligands.

26

New in BioCyc: Using Object Groups to Query, Analyze and Share Data

Michael Travers, Ingrid M. Keseler, Peter D. Karp SRI International, Artificial Intelligence Center, Menlo Park, CA 94025

BioCyc is a web-accessible collection of nearly 1700 organism-specific pathway/genome databases, mostly bacterial; the collection includes MetaCyc (MetaCyc.org), a collection of experimentally elucidated metabolic pathways, and EcoCyc (EcoCyc.org), the model organism database for Escherichia coli K-12 MG1655. Curators update the functions of individual E. coli gene products in EcoCyc based on information found in the experimental literature. The Pathway Tools software is used to create, edit and publish all BioCyc databases. The capabilities of Pathway Tools are continuously updated and improved. The BioCyc user community is encouraged to provide feedback on the functionalities of Pathway Tools and to suggest new software modules that would be useful to biologists. “Groups” is new software module that was developed as a result of a user suggestion; it can now be accessed via the Tools menu on the BioCyc web site. A group is simply a collection of objects. Individual groups can be private to the user, or can be shared with selected collaborators or all users of BioCyc. Groups can be created in several ways, including from a search output, by importing a file, and by hand. For example, a group can be created by searching EcoCyc for “rut” and selecting the group of genes that contain “rut” as part of their gene names. A gene expression data file can be imported and converted to a group; the software will enable mapping of gene names to database objects in the selected database, as well as filtering by criteria such as expression levels. The Groups tool provides several ways of examining a group of objects. A set of operations, called “transforms”, can, for example, turn a list of genes into a list of transcriptional regulators of that set of genes. There are a number of transformation operations available. The objects generated by the transformation can then be turned into a new group themselves, and similarly transformed or otherwise manipulated. For each column of database objects, properties of these objects can be added as new columns. For example, for each gene, gene name synonyms and the centisome position of the gene on the chromosome can be added. Other possible operations for groups include enrichment analyses. Enrichment analysis is a technique for identifying attributes that are statistically over- or underrepresented in a group. For example, for a set of significantly upregulated genes in a gene expression experiment, enrichment analysis can identify GO term annotations indicating biological functions or processes (e.g. “cell division”) that are statistically over-represented in that group. The user can set a desired p-value and choose from several standard statistical methods and corrections.

Further recent extensions to the Pathway Tools software have enabled systematic improvements to the E. coli metabolic model present in EcoCyc. A new Pathway Tools module that facilitates the generation of flux-balance analysis (FBA) models of metabolism was tested using EcoCyc, and a working FBA model for E. coli metabolism was generated. FBA models can be used to predict cellular growth phenotypes in varying nutrient conditions and with gene knock-outs. EcoCyc now contains a set of laboratory growth media for E. coli. For a subset of these media, data on the viability of E. coli knockout mutants is now displayed on individual gene/protein pages. This data will enable more detailed modeling of E. coli metabolism.

27

Peptidoglycan Hydrolases of Mycobacterium tuberculosis

Daniil M. Prigozhin, Daniela Mavrici, John Huizar, Hilary Vansell, and Tom Alber

Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, CA 94720

The worldwide TB epidemic compounded by the rapid rise of antibiotic resistance creates an urgent need for new therapeutics. Although potent antibiotics target the Mycobacterium tuberculosis cell wall, surprisingly little is known about how this complex structure is built and degraded. Peptidoglycan, a continuous sugar-peptide polymer that surrounds each bacterial cell, forms the foundation on which the rest of the mycobacterial cell wall is constructed. The actions of peptidoglycan hydrolases are required for growth of the bacilli and for cell division, but have to be tightly regulated to prevent self-lysis. To investigate the molecular mechanisms underpinning this delicate balance, we have identified and cloned 22 mycobacterial peptidoglycan hydrolases. Ongoing experiments aim to provide detailed biochemical and structural characterization of the recombinant proteins. As an example of this approach, we present the analysis of a newly identified N-acetylmuramoyl - L-alanine amidase, Rv3717.

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Proximal GI tract microbiome in very low birth weight neonates Imke Schröder1, Meena Garg2, Ivan Vuletic1, Jeffery F Miller1, and Vladana Milisavljevic2

1Department of Microbiology, Immunology and Molecular Genetics and 2 Division of Neonatology & Developmental Biology, Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California While transitioning from a relatively sterile intrauterine environment, neonates become colonized with a complex microbial population. Common environmental exposures influencing colonization include the maternal vaginal, fecal, or skin microflora, as well as breast milk or formula feedings, and result in the development of the intestinal microbiome. In contrast to healthy babies, preterm infants are, throughout their stay in the neonatal intensive care unit (NICU), exposed to microorganisms specific to the hospital environment. Very low birth weight (VLBW) infants (birth weight <1500g) frequently experience hospital-acquired infection (HAI) during prolonged hospitalization, which significantly impacts their mortality and morbidity. The GI microflora derived from stool samples of hospitalized, preterm neonates was shown to be different from that of healthy, full-term neonates. Current thinking is that in neonates bacteria may translocate to systemic organs and tissues increasing the risk for systemic infections, and that potentially pathogenic bacteria are more likely to translocate. However, the exact mechanisms of bacterial translocation are still not well understood. The objective of this study is to evaluate the prospective pattern of early acquisition of the proximal GI tract microbiome in VLBW neonates using 16S rDNA analysis. All subjects enrolled in this study were preterm neonates < 32 week gestational age with a birth weight <1500g. They were born within a six-month period and admitted to either the Ronald Reagan UCLA or the Santa Monica UCLA Medical Center NICUs. All neonates were enrolled in the study on day 1 of life. Serial upper GI aspirates were collected for the first 28 days of life and analyzed by week of life. Bacteria were detected in 9 of the 12 neonates using 16S rDNA sequence analysis. The proximal GI tract of VLBW infants is relatively sterile at birth and is colonized in the first four weeks of life by a phylogenetically diverse microflora of low individual complexity. By the fourth week of life, the proximal GI microbiome of the VLBW infants changed to the predominance of Firmicutes and Proteobacteria. Bacteria from both phyla are strongly implicated as causes of hospital-acquired infections.

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Genome Mining for the Discovery of Novel Nitroaromatic Antibiotics

Kou-San Ju and William Metcalf Institute for Genomic Biology, University of Illinois, Urbana, IL

Microbially synthesized nitroaromatic compounds are a promising source of new drugs and medicines to combat the continued rise of drug-resistant pathogens and cancer worldwide. Nitroaromatic natural products are comprised of chemically diverse polyketides, peptides, and heterocyclic compounds. These compounds have many useful biological properties as enzyme inhibitors, immunosuppressants, and also as anti-bacterial, -fungal, -malarial, and -cancer antibiotics. Despite their utility, nitroaromatic natural products remain understudied, with only a limited number of compounds and biosynthetic pathways having been described to date. In an effort to discover new nitroaromatic antibiotics, a sequence-guided strategy was developed to rapidly identify strains containing biosynthetic pathways for the nitroaromatic functional group. High-throughput screening of an arrayed genomic DNA library isolated from the USDA-ARS actinomycete collection and Illumina sequencing of selected strains revealed an unexpected diversity within these pathways. Ten percent of all actinomycetes contain genes for the synthesis of nitroaromatic compounds, the vast majority encoding for unknown natural products. Using different growth media, 50% of all candidate strains produced at least one nitroaromatic compound. This has led to the discovery of several promising new antibiotics, and will provide insight into the true chemical and biosynthetic diversity of this relatively unexplored group of natural products.

30

Investigating the role of Phr peptide signaling in regulating virulence in S. pneumoniae

Sharon E. Hoover1, Ho-Ching T. Tsui2, Kyle J. Wayne2, Malcolm E. Winkler2, and Beth A. Lazazzera1

1Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095

2Department of Biology, Indiana University Bloomington, Bloomington, Indiana 47405 Quorum sensing is the process by which bacteria monitor their cell density and is mediated by small secreted peptides in Gram-positive bacteria that diffuse through the environment. One family of such secreted peptides is the Phr family, which are small, unmodified peptides that are reimported by an oligopeptide permease after secretion and interact with cytoplasmic receptors to control gene expression. Phr peptides are encoded by small genes found adjacent to the genes for their cytoplasmic receptors. These Phr peptide signaling cassettes have only been characterized in Bacilli species where they regulate many important processes like virulence and sporulation. To determine whether non-Bacilli strains also produce Phr peptides for quorum sensing, we carried out a limited bioinformatic screen for genes that potentially encode Phr peptides across sequenced bacterial genomes, yielding 3 putative Phr peptide signaling modules in Streptococcus pneumoniae. One of these potential Phr peptide modules is conserved in all S. pneumoniae strains and two are found within pathogenicity islands in a subset of strains. To begin to address whether S. pneumoniae cells utilize Phr peptides for quorum sensing and the physiological role for Phr peptides in this organism, we constructed mutants that lack one of the cytoplasmic receptor proteins, tprA or it cognate Phr peptide gene, phrA. We have identified conditions under which the TprA protein is active to control gene expression, as a deletion of tprA resulted in a dramatic increase in phrA expression. These data further indicate that PhrA may autoregulate its own expression through the TprA protein. Preliminary virulence studies in a murine model for infection also indicate that the PhrA peptide may contribute to pathogenesis in S. pneumoniae. These results hint that Phr peptide signaling may not be limited to Bacilli species and provide a potential new target for antimicrobial therapies to combat pneumococcal disease.

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Metagenomic Sigma-Promoter Diversity for Genome-wide Prediction and Genetic Engineering Virgil A. Rhodius1, Kevin Clancy2, Todd Peterson2, Carol Gross1, and Christopher Voigt3. 1University of California San Francisco, 2Life Technologies, 3Massachussetts Institute of Technology. The explosion of microbial genome sequences provides a wealth of resources for discovery, enabling the identification of fundamental principles across genomes and also providing a metagenomic library of components for synthetic biology. Relating DNA sequence to transcriptional output remains a substantial problem in biology. In particular, accurately deciphering promoters from genomic sequence and forward-engineering promoter systems for controlled regulation of genetic circuits in synthetic biology are extremely difficult. We employed a metagenomic approach to decode promoters and generate tools for synthetic biology. Promoter specificity is determined by the sigma subunit of RNA polymerase. The most diverse promoters are targeted by the Group 4 sigmas, which are the most diverse and abundant of all sigmas, and regulate many key stress and developmental processes. Using synthetic metagenomics we have constructed a library of 86 Group 4 sigmas that represents their phylogenetic diversity. Using computational and experimental methods we show that the sigmas target very specific and distinct promoter sequences. This has two significant implications: 1) this enables organisms with multiple group 4 sigmas to independently control separate stress circuits; 2) this provides an ideal resource for constructing orthogonal sigma-promoter systems for sequential and parallel control of genetic modules in synthetic biology.

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Estimated Aggregation Fate revealed by Coarsening Model Fatmagül Bahar, Phillip Pratt-Szeliga and Roy D. Welch Syracuse University Department of Biology, Syracuse, NY 13244 When starved on an agar surface, a swarm containing millions of the bacterium Myxococcus xanthus undergoes a synchronized multicellular process of self-organization called development, during which sub-populations of thousands aggregate to form macroscopic mushroom-shaped structures that mature into fruiting bodies over approximately 48 hours. Across the swarm the number of aggregates does not increase steadily during development. Instead, there is a rapid increase in the number of aggregates during the first few hours, after which the number actually decreases. The arrangement of aggregates on the agar surface also changes during this process; it is random at first during the rapid increase, and then becomes more ordered as the number of aggregates decreases. The previously proposed “Traffic Jam Model”, where cells accumulate because they stop moving when the encounter an aggregate, is not capable of producing the decrease in aggregate number or increase in aggregate order. We have developed an alternative model based on the property of coarsening on thin liquid films. According to this “coarsening” model, smaller aggregates have higher free energy because their total phase boundaries are larger in relation to their volumes. In order to reduce this energy, cells within these smaller aggregates are more likely to disperse and unite with the more stable larger aggregates. In other words, small aggregates are more likely to become smaller, and larger aggregates are more likely to become larger. In this study, we report on the analysis of M. xanthus swarm development time-lapse microcinematography image stacks, and compare these results to a coarsening model whose parameters are optimized through the application of an evolutionary algorithm. After optimization, the model was able to predict the behavior of aggregates with approximately 90% accuracy.

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Phenotypic profiling of ABC transporters in Myxococcus xanthus Jinyuan Yan, Michael Bradley, and Roy D. Welch

Syracuse Univerisity, Syracuse, NY 13244

Phenotypic profiling seeks to define the structure of an organism's phenotype as a means

of uncovering molecular interactions. For example, in the bacteria Escherichia coli and

Pseudomonas aeruginosa, there are large collections of growth phenotypes under different

stresses and nutrient conditions. In our study, we characterize Myxococcus xanthus growth,

motility, and self-organization by examining the disruption of each member of the ATP-binding

cassette (ABC) transporter family. ABC transporters are ubiquitous among prokaryotes and

eukaryotes. The core components of ABC transporters include two nucleotide binding domains

(NBDs) and two transmembrane domains (TMDs). For importers, there is usually another

functional subunit called substrate binding protein (SBP). These components are located in

different polypeptides which sometimes can fuse together. ABC transporters uptake or extrude a

broad spectrum of substrates into or out from the cytoplasm, and they are thus associated with

many different cellular processes. Sometimes, members also contain extra domains which can

produce functions other than transport.

We identified 192 open reading frames (ORFs) coding for ABC transporter components

in M. xanthus genome, and produced disruption-mutation strains for each of them. After

disrupting an ORF, we characterized its mutant strain using three assays: a swarm assay, an

aggregation assay and a spore assay, all of which are tightly connected to growth, motility, and

multicellular behavior. Using a statistical analysis, we assigned different levels to mutant

phenotypes compared to wild-type, and identified connections among phenotypes and clustered

mutants according to their phenotypes. This research extends our knowledge of the nature of the

M. xanthus mutlicellular phenotype, and also reveals new connections between genotype and

phenotype.

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Morphological and microbiological characterization of Leptolyngba sp. coniform structures grown on a microbial mat Hope A. Johnson1, Kristina Reyes1, Nicolas Gonzalez1, Dickie Nguyen1, David Cho1, Nahal Ghahremani1, Frank Ospino1, John Spear2, and Joshua Stewart1 1CSU Fullerton,2Colorado School of Mines Stromatolites provide some of the earliest evidence for life on Earth. The mechanism of formation of these organosedimentary structures is unknown. Modern coniform structures found on the surface of microbial mats have similar features to stromatolites. They display a vertical element of growth and can be lithified. Molecular phylogenetic characterization of the microbial community of a coniform structure from Octopus Spring, Yellowstone National Park revealed a community dominated by Leptolyngbya sp. with heterotrophs and other cyanobacteria present in much lower abundance. The Leptolyngbya sp. was isolated from the coniform structure as well as 11 heterotrophs. Although the isolated heterotrophs were not found in the 16S rRNA gene clone library, close relatives of many of the isolated heterotrophs have been identified in association with cyanobacteria. Most of the isolates were capable of EPS production. In contrast, only the Leptolyngbya sp. and a Staphylococcus sp. isolate were motile. Motility and EPS production are traits hypothesized to be involved in cone formation. Coniform structures were cultivated in the laboratory and several factors were identified that can influence coniform morphology. These factors may have also influenced the morphology of stromatolites on early Earth.

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Bacterial RNA thermometers – The good ones and the bad ones Franz Narberhaus Microbial Biology, Ruhr University Bochum, Germany The expression of many bacterial mRNAs is controlled by the formation of complex structures in their 5’-untranslated region (5’-UTR). Riboswitches and RNA thermometers are such built-in sensory elements that control the fate of mRNAs in response to environmental conditions. Both are comprised of complex RNA structures that undergo a conformational change when a certain chemical or physical signal is present. It has been proposed that these RNA-only elements originate from an ancient RNA world. Typical RNA thermometers control translation initiation of heat shock or virulence genes by forming a secondary structure that traps the ribosome binding site (RBS). An increase in temperature to 37°C (virulence genes) or higher (heat shock genes) destabilizes the structure, liberates the RBS and permits formation of the translation initiation complex. The presentation will address the following questions: (i) What are the requirements for a functional RNA thermometer and what are the molecular details of RNA melting? (ii) Is melting of the RNA reversible and is this process physiologically relevant? (iii) Do RNA thermometers contribute to bacterial fitness under heat stress conditions and to virulence in bacterial pathogens? References Chowdhury S, Maris C, Allain FH, Narberhaus F (2006) Molecular basis for temperature sensing

by an RNA thermometer. EMBO J., 25:2487-2497. Waldminghaus T, Heidrich N, Brantl S, Narberhaus, F (2007) FourU: a novel type of RNA

thermometer in Salmonella. Mol. Microbiol., 65:413-424. Kortmann J, Sczodrok S, Rinnenthal,J, Schwalbe H, Narberhaus F (2011) Translation on

demand by a simple RNA-based thermosensor. Nucleic Acids Res. 39:2855-2868. Kortmann J, Narberhaus F (2012) Bacterial RNA thermometers: Molecular zippers and

switches. Nat. Rev. Microbiol., in press.

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Progress in Understanding Predatory Behavior of Ensifer adhaerens toward Micrococcus luteus. Mark O. Martin*, Tameka Smith, and Jared Trecker Department of Biology, University of Puget Sound 1500 N. Warner Street #1088, Tacoma, WA 98416 E-mail: momartin@pugetsound.edu Telephone: 253-879-2747 Ensifer adhaerens, a predator of Micrococcus luteus, was originally found in soil samples by Lester Casida during the 1970s. On low nutrient medium plates, Ensifer displays a predatory behavior termed “tracking,” in which the bacterium follows a track of Micrococcus cells while generating copious exopolysaccharides. This behavior does not occur with killed Micrococcus luteus, culture supernatants, or with related bacterial species. 16s rRNA analysis has shown that E. adhaerens is an alpha proteobacter, and closely related to fast growing rhizobia (such as Sinorhizobium fredii), but does not possess nod or nif genes. Little is yet known of potential alternative prey species, recognition mechanisms, or details of this predatory behavior by Ensifer. Though the organism grows well under laboratory conditions, high resistance to a number of antibiotics has made typical molecular genetic approaches to studying this organism a challenge. As a first step to studying this organism, EMS mutagenesis was used to identify mutants of E. adhaerens defective in producing exopolysaccharide; two such mutants were isolated. Both were shown to be capable of tracking along streaks of M. luteus on low nutrient medium, demonstrating that the exopolysaccharide was not necessary for this predatory behavior. Some progress has also been made with transposon mutagenesis using a spectinomycin resistant element; sites of insertion were “self-cloned” and DNA analysis revealed seemingly random sites of insertion and surrounding sequence typical of fast growing rhizobia. Thus, we believe that transposon mutagenesis is now a more practicable tool for the study of Ensifer adhaerens. Current research is focused on generating motility defective mutants of Ensifer (to evaluate a possible role in tracking) and attempting to design more direct screens for defects in predatory behavior. We are also interested in learning more about the genome of this understudied bacterium and comparing it to better known fast growing rhizobia. It may well be that many soil bacteria have predatory behaviors under the proper conditions and with the correct prey organisms, which could in turn impact the population structure and diversity of soil microbiota.

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Michele Igo, Department of Microbiology, UC Davis Autotransporters: Secreted proteins important for Xylella fastidiosa virulence