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Recent Publications
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Wall, J.D., Arkin, A.P., Balci, N.C. and Rapp-Giles, B. (2008) Genetics and genomics of sulfate respiration in Desulfovibrio. Springer-Verlag, Berlin, Heidelberg, Microbial Sulfur Metabolism(1):1-12 [View the Publication]
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Abstract
Bacteria that have evolved to use sulfate as a terminal electron acceptor must commit to spending energy for sulfate activation before there is a return on the investment allowing net energy gain. How sulfate is used and how electron flow
is controlled have provided challenging topics for research for many years. Having the complete genome sequences of several of these bacteria is a monumental step in the elucidation of these questions. This information has provided the tools for determining the quantity of transcripts for genes under defined growth conditions, not just the relative changes in transcripts in two growth conditions. A comparison of the hybridization signal of messenger RNA with that of genomic DNA with oligonucleotide microarrays of all open reading frames reveals the differences in steady-state levels of transcripts for each gene. Growth of Desulfovibrio vulgaris Hildenborough on defined medium with lactate as a carbon and reductant source and with sulfate as the electron acceptor has been examined by this procedure for levels of gene expression. Relative functional importance was inferred from the levels of gene transcription, in spite of the recognized limitations of this interpretation. Not surprisingly, genes encoding established functions for sulfate reduction were highly expressed. However, the high molecular mass c-type cytochrome genes thought to
encode a most important transmembrane electron conduit for sulfate reduction were expressed at quite low levels. Funding Source
ESPP Keywords
Comparative Genomics, Microarrays, Sequencing, Sulfate Reducers, Transcriptomics |
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Gaucher, Sara P.; Redding, Alyssa M.; Mukhopadhyay, Aindrila; Keasling, Jay D. and Singh, Anup K. (2008) Post-Translational Modifications of Desulfovibrio vulgaris Hildenborough Sulfate Reduction Pathway Proteins. J. Proteome Res., : [View the Publication]
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Abstract
Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications (PTMs). Although PTMs are a critical aspect of cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase γ-subunit (DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium, Desulfovibrio desulfuricans G20, also showed similar +42 Da modifications in the same pathway. Here, we discuss our methods and implications of potential trimethylation in the D. vulgaris sulfate reduction pathway. Funding Source
ESPP, Metabolomics Keywords
Functional Genomics, Metabolomics, Proteomics, Sulfate Reducers |
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Recent Presentations
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Stolyar, S.; Pinel, N.; Walker, C. B.; Wall, J.; Stahl, D.A. The physiological role of the cytoplasmic hydrogenases in D. vulgaris Hildenborough, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology
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Abstract
The Gram-negative Deltaproteobacterium D. vulgaris Hildenborough is able to grow with sulfate, sulfite and thiosulfate as electron acceptors and in their absence via fermentation or syntrophic association with hydrogenotrophic organisms. Despite decades of research, the mechanism of energy generation by D. vulgaris is not well understood. Genome sequence revealed genes for at list six different hydrogenases, four periplasmic and two cytoplasmic. Although some of them have been characterized, their roles in D. vulgaris remain obscure. We have examined the consequences of mutations in two cytoplasmic hydrogenases on respiratory and syntrophic growth: 1) echA (DVU0434), the first gene in the operon for the Ech type NiFe- containing hydrogenase and 2) cool (DVU2288), the third gene in the operon coding for the second cytoplasmic NiFe-containing hydrogenase Coo. Growth rate, cell yield, and metabolite production were characterized for three growth conditions: i) sulfate with lactate or pyruvate, ii) sulfate with acetate and hydrogen, and iii) in syntrophic association with a hydrogenotrophic methanogen. Hydrogen oxidation activities in soluble and membrane fractions of the mutants and a wild type were not significantly different. Although growth rates of both mutants on sulfate with pyruvate or lactate were comparable to the wild type, hydrogen evolution was much greater for the echA mutant. Growth of the echA mutant was severely impaired relative to the wild type or cooL mutant with sulfate and hydrogen/CO2, but this mutation had little affect on syntrophic growth on either lactate or pyruvate. Syntrophic growth of the cooL mutant was severely impaired on lactate but not on pyruvate. Based on these observations we concluded that the main role of the Ech hydrogenase is in hydrogen oxidation. The Coo hydrogenase likely directly coupled the oxidation of lactate to pyruvate by accepting electrons and reducing protons inside the cells. Presenter
Stolyar, S. Funding Source
Environmental Stress Pathway Project (ESPP) Keywords
Comparative Genomics, Environmental Genomics, Evolutionary Biology, Extremophiles, Sulfate Reducers |
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Materna, A.C.; Clarke, S.A.; Cruz, C.; Gao, X.; Alm, E.J. Natural Diversity and Experimental Evolution of Environmental Stress Tolerance in Marine Bacteria, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology
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Abstract
Genome sequencing has revealed extensive genetic variation within bacterial species and among co-existing bacteria. Using marine Vibrio strains as a model system, we investigate to what extent observed sequence diversity corresponds to measurable differences in salinity and temperature tolerance phenotypes, two ecologically important factors for this group of organisms. Using directed evolution, we quantify how malleable these phenotypes are with respect to a small number of mutation events. We have designed two-dimensional gradients in 24 cm square dishes containing solid growth medium to monitor temperature and salinity tolerances over a broad range of both factors. Growth patterns indicate the strain-specific minimum and maximum tolerances and interactions between the factors (salinity and temperature). We compared the specific boundaries of growth for multiple strains of Vibrio splendidus and V. alginolyticus. While the obtained profiles differ in their shape and limits, some consistent features appear. Tolerance to increasing salinities correlated positively with temperature tolerance. However, higher salinity constrained the limits of temperature tolerance, so that the maximum salinity tolerance occurred at intermediate temperatures. Similarly, growth at higher temperatures led to a tradeoff, limiting the range of salinity tolerance. Interestingly, at high salinities, low temperatures tended to suspend growth, leaving viable cells that could be regenerated when the temperature gradient was removed, while higher temperatures led to killing. In addition to comparing related environmental isolates, this method was further applied to study differences between parental and evolved strains. Serial application of 106 cells/cm2 to the solid medium gradients enabled selection for spontaneous, more tolerant mutants. In future work, this integrated ecological and experimental approach will be combined with genome re-sequencing to draw connections between genetic diversity and ecologically relevant phenotypes and tradeoffs. Presenter
Materna, A.C. Funding Source
Environmental Stress Pathway Project (ESPP) Keywords
Environmental Genomics, Evolutionary Biology, Extremophiles, Models, Sequencing, Stress Response |
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