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109 publications were found
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Kazakov, Alexei E., Cipriano, Michael J., Novichkov, Pavel S., Minovitsky, Simon, Vinogradov, Dmitry V., Arkin, Adam P., Mironov, Andrey A., Gelfand, Mikhail S., and Dubchak, Inna (2007) RegTransBase - a database of regulatory sequences and interactions in a wide range of prokaryotic genomes. Nucleic Acids Research, 35(Database Issue): D407-D412 [View the Publication]closeAbstractRegTransBase, a manually curated database of regulatory interactions in prokaryotes, captures the knowledge in published scientific literature using a controlled vocabulary. Although a number of databases describing interactions between regulatory proteins and their binding sites are currently being maintained, they focus mostly on the model organisms Escherichia coli and Bacillus subtilis, or are entirely computationally derived. RegTransBase describes a large number of regulatory interactions reported in many organisms and contains various types of experimental data, in particular: the activation or repression of transcription by an identified direct regulator; determining the transcriptional regulatory function of a protein (or RNA) directly binding to DNA (RNA); mapping or prediction of binding site for a regulatory protein; characterization of regulatory mutations.
Currently, the RegTransBase content is derived from about 3000 relevant articles describing over 7000 experiments in relation to 128 microbes. It contains data on the regulation of about 7500 genes and evidence for 6500 interactions with 650 regulators. RegTransBase also contains manually created position weight matrices (PWM) that can be used to identify candidate regulatory sites in over 60 species. RegTransBase is available at http://regtransbase.lbl.gov
Funding SourceESPP KeywordsBioinformatics, Comparative Genomics |
| 22 |
Mukhopadhyay, Aindrila, Redding, Alyssa M., Joachimiak, Marcin P., Arkin, Adam P., Borglin, Sharon E., Dehal, Paramvir S., Chakraborty, Romy, Geller, Jil T.,Hazen, Terry C., He, Qiang, Joyner, Dominique C.,Martin, Vincent J. J., Wall, Judy D., Yang, Zamin Koo, Zhou, Jizhong and Keasling, Jay D. (2007) Cell wide responses to low oxygen exposure in Desulfovibrio vulgaris Hildenborough. J. Bacteriol., 189(16):5996-6010 [View the Publication]closeAbstractThe responses of the anaerobic, sulfate-reducing Desulfovibrio vulgaris Hildenborough to low oxygen exposure (0.1% O2) were monitored via transcriptomics and proteomics. Exposure to 0.1% O2 caused a decrease in growth rate without affecting viability. A concerted up-regulation in the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O2 exposure. Several of these candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c3 complex. Other known oxidative stress response candidates remained unchanged during this low O2 exposure. To fully understand the results of the 0.1% O2 exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O2 exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were down regulated during air exposure. Our results highlight the differences in the cell wide response to low and high O2 levels of in D. vulgaris and suggest that while exposure to air ishighly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O2 levels in its environment.The responses of the anaerobic, sulfate-reducing Desulfovibrio vulgaris Hildenborough to low oxygen exposure (0.1% O2) were monitored via transcriptomics and proteomics. Exposure to 0.1% O2 caused a decrease in growth rate without affecting viability. A concerted up-regulation in the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O2 exposure. Several of these candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c3 complex. Other known oxidative stress response candidates remained unchanged during this low O2 exposure. To fully understand the results of the 0.1% O2 exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O2 exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were down regulated during air exposure. Our results highlight the differences in the cell wide response to low and high O2 levels of in D. vulgaris and suggest that while exposure to air ishighly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O2 levels in its environment. Funding SourceESPP, Metabolomics, PCAP KeywordsFunctional Genomics, Proteomics, Stress Response, Sulfate Reducers, Transcriptomics |
| 23 |
Pingitore, Francesco, Tang, Yinjie, Kruppa, Gary H. and Keasling, Jay D. (2007) FT-ICR MS: a new tool for isotopomer analysis in amino acids. Analytical Chemistry, 79(6):2483-2490 [View the Publication]closeAbstractFluxes through known metabolic pathways and the presence of novel metabolic reactions are often determined by feeding isotopically labeled substrate to an organism and then determining the isotopomer distribution in amino acids in proteins. However, commonly used techniques to measure the isotopomer distributions require derivatization prior to analysis (gas chromatography/mass
spectrometry (GC/MS)) or large sample sizes (nuclear magnetic resonance (NMR) spectroscopy). Here, we demonstrate the use of Fourier transform-ion cyclotron resonance mass spectrometry with direct infusion via electrospray ionization to rapidly measure the amino acid isotopomer distribution in a biomass hydrolysate of the soil bacterium Desulfovibrio vulgaris Hildenborough.
By applying high front-end resolution for the precursor ion selection followed by sustained off-resonance irradiation collision-induced dissociation, it was possible to determine exactly and unambiguously the specific locations
of the labeled atoms in the amino acids, which usually requires a combination of 2-D 13C NMR spectroscopy and GC/MS. This method should be generally
applicable to all biomass samples and will allow more accurate determination of metabolic fluxes with less work and less sample. Funding SourceESPP KeywordsExtremophiles, Functional Genomics, Metabolomics, Proteomics |
| 24 |
Podar, Mircea, Abulencia, Carl B., Walcher, Marion, Hutchison, Don, Zengler, Karsten, Garcia, Joseph A., Holland, Trevin, Cotton, David, Hauser, Lore and Keller, Martin (2007) Targeted Access to the Genomes of Low Abundance Organisms in Complex Microbial Communities. (In Press)closeAbstractCurrent metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism, and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequency, especially those representing yet uncultured taxa--which includes the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms directly isolated from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community metabolic potential. Funding SourceESPP KeywordsEnvironmental Genomics, Field Studies, Functional Genomics, Metagenomics |
| 25 |
Price, M.N., Dehal, P.S. and Arkin, A.P. (2007) Horizontal gene transfer and the evolution of transcriptional regulation in Escherichia coli. Genome Biology, 9(1):R4 [View the Publication]closeAbstractBackground: Most bacterial genes were acquired by horizontal gene transfer from other bacteria instead of being inherited by continuous vertical descent from an ancient ancestor}. To understand how the regulation of these {acquired} genes evolved, we examined the evolutionary histories of transcription factors and of regulatory interactions from the model bacterium Escherichia coli K12. Results: Although most transcription factors have paralogs, these usually arose by horizontal gene transfer rather than by duplication within the E. coli lineage, as previously believed. In general, most neighbor regulators -- regulators that are adjacent to genes that they regulate -- were acquired by horizontal gene transfer, while most global regulators evolved vertically within the gamma-Proteobacteria. Neighbor regulators were often acquired together with the adjacent operon that they regulate, so the proximity might be maintained by repeated transfers (like "selfish operons"). Many of the as-yet-uncharacterized (putative) regulators have also been acquired together with adjacent genes, so we predict that these are neighbor regulators as well. When we analyzed the histories of regulatory interactions, we found that the evolution of regulation by duplication was rare, and surprisingly, many of the regulatory interactions that are shared between paralogs result from convergent evolution. Another surprise was that horizontally transferred genes are more likely than other genes to be regulated by multiple regulators, and most of this complex regulation probably evolved after the transfer. Conclusions: Our results highlight the rapid evolution of niche-specific gene regulation in bacteria. Funding SourceESPP KeywordsEvolutionary Biology, Transcriptomics |
| 26 |
Price, Morgan N., Dehal, Paramvir S., Arkin, Adam P. (2007) Orthologous Transcription Factors in Bacteria Have Different Functions and Regulate Different Genes. PLoS Computational Biology, 3(9):e175 [View the Publication]closeAbstractTranscription factors (TFs) form large paralogous gene families and have complex evolutionary histories. Here, we ask whether putative orthologs of TFs, from bidirectional best BLAST hits (BBHs), are evolutionary orthologs with
conserved functions. We show that BBHs of TFs from distantly related bacteria are usually not evolutionary orthologs. Furthermore, the false orthologs usually respond to different signals and regulate distinct pathways, while the few
BBHs that are evolutionary orthologs do have conserved functions. To test the conservation of regulatory interactions, we analyze expression patterns. We find that regulatory relationships between TFs and their regulated genes are
usually not conserved for BBHs in Escherichia coli K12 and Bacillus subtilis. Even in the much more closely related bacteria Vibrio cholerae and Shewanella oneidensis MR-1, predicting regulation from E. coli BBHs has high error rates.
Using gene–regulon correlations, we identify genes whose expression pattern differs between E. coli and S. oneidensis. Using literature searches and sequence analysis, we show that these changes in expression patterns reflect changes in gene regulation, even for evolutionary orthologs. We conclude that the evolution of bacterial regulation should be analyzed with phylogenetic trees, rather than BBHs, and that bacterial regulatory networks evolve more rapidly than
previously thought. Funding SourceESPP KeywordsBioinformatics, Comparative Genomics, Sequencing, Transcriptomics |
| 27 |
Shaikh, Afshan S., Tang, Yinjie J., Mukhopadhyay, Aindrila, Keasling, Jay D. (2007) Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein. (In Press)closeAbstract13C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogenous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities. Funding SourceESPP, Metabolomics, PCAP KeywordsComparative Genomics, Functional Genomics, Metabolomics, Proteomics |
| 28 |
Stolyar, Sergey, Van Dien, Steve, Hillesland, Kristina L., Pinel, Nicolas, Lie, Thomas J., Leigh, John A., and Stahl, David A. (2007) Metabolic modeling of a mutualistic microbial community. Molecular Systems Biology, 3:92 [View the Publication]closeAbstractThe rate of production of methane in many environments depends upon mutualistic interactions between sulfate-reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co-culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities. Funding SourceESPP KeywordsComparative Genomics, Environmental Genomics, Field Studies, Metabolism, Models, Sulfate Reducers |
| 29 |
Tang, Y.J. and Krieger-Brockett, B. (2007) Mathematical analysis of the whole core injection method accuracy for measuring phenanthrene biodegradation rates in undisturbed marine sediments. Chemosphere, Epub ahead of print: [View the Publication]closeAbstractRates of 14C-phenanthrene mineralization in contaminated, undisturbed marine sediments were measured using the whole core injection method to assess microbial natural attenuation activity as a function of sediment depth. Submerged sediments were sampled from Eagle Harbor, a marine superfund site in Puget Sound. Experiments show significant biodegradation activities (0.0012–0.0036 day−1) in the sediment horizons from 0 to 10 cm. The purpose and scope of this paper is to evaluate the range of experimental conditions giving valid results; a mathematical simulation described competing contaminant 14C-phenanthrene diffusion and simultaneous biodegradation (Monod kinetics), both retarded by sorption. The effect of aging was examined with two sorption models in presumed pseudo-homogenous sediments having effective properties. The simulation predictions provide quantitative guidelines for the successful use of the whole core injection method. (1) The effective Monod constant Click to view the MathML source in sediment is increased by a large partition coefficient KP between sediment and water and makes the apparent 14C-phenanthrene biodegradation approach first-order kinetics. (2) When Click to view the MathML source, the measured 14C-phenanthrene biodegradation extent is biased by inadequately distributed injected tracer only when less than 7% of the sediment horizon is initially probed and mixed with injected tracer. (3) A short incubation time (<20 days) is necessary when a mobile indicator, e.g., gaseous 14CO2, is used. For longer incubation times, predictions show that a 14CO2 indicator diffuses to adjacent horizons, thus smearing the depth profile of biodegradation. (4) This method employing a radiolabeled tracer provides accurate biodegradation rates for freshly contaminated sediments, and represents an upper limit to the natural phenanthrene biodegradation extents if the contaminant is aged over 50 days. Funding SourceESPP KeywordsBioremediation, Metabolomics |
| 30 |
Tang, Yinjie J., Judy S. Hwang, David E. Wemmer, and Jay D. Keasling (2007) Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions. Applied and Environmental Microbiology, 73(3):718-729 [View the Publication]closeAbstractThe central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values. Funding SourceESPP, Metabolomics KeywordsFunctional Genomics, Metabolomics, Stress Response |
| 31 |
Tang, Yinjie J., Adam L. Meadows, James Kirby, and Jay D. Keasling (2007) Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling. Journal of Bacteriology, 189(3):894-901 [View the Publication]closeAbstractIt has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions. Funding SourceESPP, Metabolomics KeywordsFunctional Genomics, Metabolomics, Stress Response |
| 32 |
Tang, Yinjie J., Chakraborty, Romy, Martin, Hector Garcia, Chu, Jeannie, Hazen, Terry C., Keasling, Jay D. (2007) Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid. Applied and Environmental Microbiology, 73(12):3859-3864 [View the Publication]closeAbstractWe analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens. Funding SourceMetabolomics KeywordsFunctional Genomics, Metabolomics, Stress Response |
| 33 |
Tang, Yinjie J., Jared M. Ashcroft, Ding Chen, Guangwei Min, Chul-Hyun Kim, Bipasha Murkhejee, Carolyn Larabell, Jay D. Keasling and Fanqing Frank Chen (2007) Charge-Associated Effects of Fullerene Derivatives on Microbial Structural Integrity and Central Metabolism. Nano Lett., 7(3):754 -760 [View the Publication]closeAbstractThe effects of four types of fullerene compounds (C60, C60-OH, C60-COOH, C60-NH2) were examined on two model microorganisms (Escherichia coli W3110 and Shewanella oneidensis MR-1). Positively charged C60-NH2 at concentrations as low as 10 mg/L inhibited growth and reduced substrate uptake for both microorganisms. Scanning electron microscopy (SEM) revealed damage to cellular structures. Neutrally charged C60 and C60-OH had mild negative effects on S. oneidensis MR-1, whereas the negatively charged C60-COOH did not affect either microorganism's growth. The effect of fullerene compounds on global metabolism was further investigated using [3-13C]L-lactate isotopic labeling, which tracks perturbations to metabolic reaction rates in bacteria by examining the change in the isotopic labeling pattern in the resulting metabolites (often amino acids).1-3 The 13C isotopomer analysis from all fullerene-exposed cultures revealed no significant differences in isotopomer distributions from unstressed cells. This result indicates that microbial central metabolism is robust to environmental stress inflicted by fullerene nanoparticles. In addition, although C60-NH2 compounds caused mechanical stress on the cell wall or membrane, both S. oneidensis MR-1 and E. coli W3110 can efficiently alleviate such stress by cell aggregation and precipitation of the toxic nanoparticles. The results presented here favor the hypothesis that fullerenes cause more membrane stress4-6 than perturbation to energy metabolism. Funding SourceESPP KeywordsMetabolomics |
| 34 |
Tang, Yinjie J., Meadows, Adam L., Keasling, Jay D. (2007) A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion. Biotechnol Bioeng., 96(1):125-133 [View the Publication]closeAbstractAerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition
across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even
at low DO levels, presumably due to a very low Km for oxygen; cells metabolize acetate (maximum specific growth rate, mmax,A of 0.28 h-1 as a single carbon source slower than they metabolize lactate (mmax,L of 0.47 h-1) and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions. Funding SourceESPP KeywordsBioremediation, Metabolomics |
| 35 |
Tang, Yinjie, Pingitore, Francesco, Mukhopadhyay, Aindrila, Phan, Richard, Hazen, Terry C. and Keasling, Jay D. (2007) Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry. J. Bacteriol., 189(3):940-949 [View the Publication]closeAbstractFlux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both GC-MS and Fourier Transform-Ion Cyclotron Resonance mass spectrometry (FT-ICR MS) indicate the lack of oxidatively functional TCA cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and the reactions involved are the primary route of energy production (NAD(P)H and ATP production). Independent of the TCA cycle, direct cleavage of acetyl-CoA to CO and 5,10-methyl-THF also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent
oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports (the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase). These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris, and also demonstrate FT-ICR MS as a powerful tool for isotopomer analysis, overcoming problems in both GC-MS and NMR spectroscopy. Funding SourceESPP, Metabolomics, PCAP KeywordsMetabolomics |
| 36 |
Van Nostrand, Joy D, Khijniak, Tatiana V., Gentry, Terry J., Novak, Michelle T., Sowder, Andrew G., Zhou, Jizhong .Z., Bertsch, Paul M. and Morris, Pamela J. (2007) Isolation and Characterization of Four Gram-Positive Nickel-Tolerant Microorganisms from Contaminated Riparian Sediments. Microb. Ecol., 53(3):670-682 [View the Publication]closeAbstractMicrobial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared to pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes. Funding SourceESPP KeywordsBioremediation, Extremophiles, Field Studies, Stress Response |
| 37 |
Wagner, M., Smidt, H., Loy, A., and Zhou, J. (2007) Unravelling Microbial Communities with DNA-Microarrays: Challenges and Future Directions. . Microb. Ecol., 53(3):498-506closeAbstractHigh-throughput technologies are urgently needed for monitoring the formidable biodiversity and functional capabilities of microorganisms in the environment. Ten years ago, DNA microarrays, miniaturized platforms for highly parallel hybridization reactions, found their way into environmental microbiology and raised great expectations among researchers in the field. In this article, we briefly summarize the state-of-the-art of microarray approaches in microbial ecology research and discuss in more detail crucial problems and promising solutions. Finally, we outline scenarios for an innovative combination of microarrays with other molecular tools for structure-function analysis of complex microbial communities. Funding SourceESPP KeywordsMicroarrays |
| 38 |
Wu, W., Carley, J., Luo, J., Ginder-Vogel, M.A., Cardenas, E., Leigh, M., Hwang, C.,
Kelly, S.D., Ruan, C., Wu, L., Gentry, T.J., Lowe, K., Mehlhorn, T., Carroll, S.L.,
Fields, M.W., Gu, B., Watson, D.B., Kemner, K.M., Marsh, T., Tiedje, J., Zhou, J.,
Fendorf, S., Kitanidis, P., Jardine, P.M., Criddle, C.S (2007) In situ bioreduction of uranium (VI) to submicromolar levels and reoxidation by dissolved oxygen. Environ. Sci. Technol. , 41(16):5716-5723 [View the Publication]closeAbstractGroundwater within Area 3 of the U.S. Department of Energy (DOE) Environmental Remediation Sciences Program (ERSP) Field Research Center at Oak Ridge, TN (ORFRC) contains up to 135 íM uranium as U(VI). Through
a series of experiments at a pilot scale test facility, we explored the lower limits of groundwater U(VI) that can be achieved by in-situ biostimulation and the effects of dissolved oxygen on immobilized uranium. Weekly 2 day additions of ethanol over a 2-year period stimulated growth of denitrifying, Fe(III)-reducing, and sulfate-reducing bacteria, and immobilization of uranium as U(IV), with dissolved uranium concentrations decreasing to low levels. Following sulfite addition to remove dissolved oxygen, aqueous U(VI) concentrations fell below the U.S.
Environmental Protection Agengy maximum contaminant limit (MCL) for drinking water (<30 íg L-1 or 0.126 íM). Under anaerobic conditions, these low concentrations were stable, even in the absence of added ethanol. However, when sulfite additions stopped, and dissolved oxygen (4.0-5.5 mg L-1) entered the injection well, spatially variable changes in aqueous U(VI) occurred over a 60 day period, with concentrations increasing rapidly from <0.13 to 2.0 íM at a multilevel sampling (MLS) well located close to the injection well, but changing little at an MLS well located further away. Resumption of ethanol addition restored reduction of Fe(III), sulfate, and U(VI) within 36 h. After 2 years of ethanol addition, X-ray absorption near-edge structure spectroscopy (XANES) analyses indicated that U(IV) comprised 60-80% of the total uranium in sediment samples. At the completion of the project (day 1260), U concentrations in MLS wells were less than 0.1 íM. The microbial community at MLS wells with low U(VI) contained bacteria that are known to reduce uranium, including Desulfovibrio spp. andGeobacter spp., in both sediment and groundwater. The dominant Fe(III)-reducing species were Geothrix spp. Funding SourceESPP, ERSP KeywordsBioremediation, Extremophiles, Field Studies |
| 39 |
Yergeau, Etienne, Kang, Sanghoon, He, Zhili, Zhou, Jizhong and Kowalchuk, George A. (2007) Functional microarray analysis of nitrogen and carbon cycling genes across an Antarctic latitudinal transect. The ISME Journal: Multidisciplinary Journal of Microbial Ecology, 1(2):163-179 [View the Publication]closeAbstractSoil-borne microbial communities were examined via a functional gene microarray approach across a southern polar latitudinal gradient to gain insight into the environmental factors steering soil N and C-cycling in terrestrial Antarctic ecosystems. The abundance and diversity of functional gene families were studied for soil-borne microbial communities inhabiting a range of environments from 511S (cool temperate – Falkland Islands) to 721S (cold rock desert – Coal Nunatak). The recently designed functional gene array used contains 24 243 oligonucleotide probes and covers >10,000 genes in >150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance and organic contaminant degradation (He et al. 2007). The detected N- and C-cycle genes were significantly different across different sampling locations and vegetation types. A number of significant trends were observed regarding the distribution of key gene families across the environments examined. For example, the relative detection of cellulose degradation genes was correlated with temperature, and microbial C-fixation genes were more present in plots principally lacking vegetation. With respect to the N-cycle, denitrification genes were linked to higher soil temperatures, and N2-fixation genes were linked to plots mainly vegetated by lichens. These microarray-based results were confirmed for a number of gene families using specific real-time PCR,
enzymatic assays and process rate measurements. The results presented demonstrate the utility of an integrated functional gene microarray approach in detecting shifts in functional community properties in environmental samples and provide insight into the forces driving important processes of terrestrial Antarctic nutrient cycling. Funding SourceESPP, ERSP KeywordsEnvironmental Genomics, Functional Genomics, Microarrays |
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Yooseph, S., Sutton, G., Rusch, D.B., Halpern, A.L., Williamson, S.J., Remington, K., Eisen, J.A., Heidelberg, K.B., Manning, G., Li, W., Jaroszewski, L., Cieplak, P.
Miller, C.S., Li, H., Mashiyama, S.T., Joachimiak, M.P., van Belle, C., Chandonia, J.M., Soergel, D.A., Zhai, Y., Natarajan, K., Lee, S., Raphael, B.J., Bafna, V., Friedman, R., Brenner, S.E., Godzik, A., Eisenberg, D., Dixon, J.E., Taylor, S.S.,
Strausberg, R.L., Frazier, M., Venter, J.C., (2007) The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families. PLoS Biol., 5(5):e16 [View the Publication]closeAbstractMetagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature. Funding SourceESPP KeywordsMetagenomics, Proteomics |
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