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327 presentations were found
| 181 |
Zhou, Aifen
Zhili He
Chris Hemme
Aindrila Mukhopadhyay
Jay Keasling
Adam P. Arkin
Terry C. Hazen
Judy D. Wall
Jizhong Zhou Global Gene Regulation in Desulfovibrio vulgaris Hildenborough, 02/12/2007, North Bethesda, MD, GTL: Genomics to Life Grantees' Workshop, [Poster-AifenZhou-final.pdf]Desulfovibrio vulgaris Hildenborough (DvH) is an obligate anaerobe and has been used as a model organism for studying the energy metabolism of sulfate-reducing bacterium (SRB). However, experimental data about the transcriptional regulatory networks which are essential for understanding the cellular processes are very limited. One of the central goals of ESPP is to link laboratory measurements of stress responses and metabolism to activities of microbes in the field. Thus, towards this goal, we have performed laboratory study of gene expression and regulations in D. vulgaris in responses to oxidative stress and the importance of key global regulatory genes in stress responses. Several predicted global regulators are investigated via mutant characterization, transcriptomic assay and their in vivo gene regulations using ChIP-chip assay.
CRP/FNR. CRP/FNR regulators are DNA binding proteins function as positive transcription factors. There are four CRP/FNR homologues in the DvH genome (DVU2547, DVU0379, DVU3111, DVU2097). Evidence from other bacteria demonstrated that CRP/FNR regulators function in response to a broad spectrum of intracellular and exogenous signals such as oxidative and nitrosative stress, nitric oxide, carbon monoxide or temperature. Microarray data from DvH shows that their transcript levels are altered in response to nitrate, nitrite, heat shock, and oxygen stresses. To determine the function to the DvH CRP/FNR, knockout mutants for all four CRP/FNR proteins were generated. The mutants will be characterized using various electron donors and acceptors, different stressors, and transcriptomic analysis. To study the global gene regulation by CRP/FNR, recombinant proteins for all four CRP/FNR were obtained and polyclonal antibodies were generated. Immunoprecipitated DNA-protein complexes with specific CRP/FNR polyclonal antibodies will be hybridized to the DvH PCR-amplicon promoter array. And the CRP/FNR binding motif can be identified by computational and experimental approaches.
H2O2 stress response. Oxidative stress is one of the most common environmental stressors. Evidences show that DvH cells are aero-tolerant although they are strict anaerobe. But little is known about molecular mechanisms of oxidative stress responses. DvH is one of the few microorganisms that contain both defense systems which are typical for the aerobic (Sod and Kat) and the anaerobic (Rub, Rbr, Rbo etc.) microbes, but their roles remain elusive. In this study, DvH cells were stressed with two different concentrations of H2O2 (1 mM, 4 mM) and 5 time-points (30, 60, 120, 240 and 480 min) were used for the transcriptomic analysis. Microarray data demonstrated that higher concentration of H2O2 had broader effect on gene expression. The time-points with the greatest gene expression changes are 120 min (485 up and 527 down) and 240 min (750 up and 753 down) for 1 mM and 4 mM of H2O2 respectively. Rdl, Rbr2 were up-regulated, which suggest that these two proteins, rather than Rub-Rbo & Rbr suggested by Coulter’s in vitro experiment data, may play major roles in H2O2 stress. Genes in the predicted PerR and FUR regulon were also up-regulated. Some interesting candidates such as DVU3269 (a hybrid histidine kinase (HK)), DVU3136 (a nitroreductase family protein) etc. were significantly up-regulated., The function of the putative candidates in H2O2 stress response are going to be confirmed by other approaches such as knockout mutant analysis.
FUR, PerR and ZUR. FUR, PerR and ZUR are Fur Paralogs in the DvH genome. Microarray data show that they are involved in iron acquisition, acid shock response and oxidative stress etc. The functional analysis of these three global transcription regulators is in progress. PresenterZhou, Aifen Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsComparative Genomics, Environmental Genomics, Metabolomics, Microarrays, Models, Sequencing, Stress Response, Sulfate Reducers, Transcriptomics |
| 182 |
Hazen*, T. C., H.-Y. N. Holman, J. Keasling, A. Mukhopadhyay, S. Chhabra, J. T. Geller, M. Singer, D. Joyner, T. Torok, J. Wall, D. Elias, and M. D. Biggin. Invited. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes During Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. , 02/12/2007, North Bethesda, MD, Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes During Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. PresenterHazen, T. C. Funding SourceProtein Complex Analysis Project (PCAP) KeywordsBiomass Production, Proteomics, Stress Response, Sulfate Reducers |
| 183 |
Hazen, T. C. and Arkin, A.P., Invited Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution, 02/10/2007, Atlanta, GA, School of Biology, Georgia Institute of Technologytbd PresenterHazen, T. C. Invited Funding SourceEnvironmental Stress Pathway Project (ESPP), Metabolomics, Protein Complex Analysis Project (PCAP), ERSP (formerly known as NABIR) KeywordsComparative Genomics, Environmental Genomics, Functional Genomics, Lipidomics, Metabolomics, Metagenomics, Phenomics, Transcriptomics |
| 184 |
Daly*, R. A., E. L. Brodie, Y. Kim, J. M. Wan, T. K. Tokunaga, G. L. Andersen, T. C. Hazen, and M. K. Firestone Contributed. Influence of Electron Donor Type and Concentration on Dynamics of Bacterial Populations Associated with Uranium Reduction and Remobilization, 02/07/2007, Toronto, Canada, Annual Meeting American Society for Microbiologytbd PresenterDaly*, R. A. Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBioremediation, Environmental Genomics, Field Studies, Stress Response |
| 185 |
Butland, Gareth, Ming Dong, Steven C. Hall, Bing K. Jap, Jian Jin, Susan J. Fisher, Peter J. Walian, H. Ewa Witkowska, Lee Yang, Mark D. Biggin Protein Complex Analysis Project (PCAP): Multi-Protein Complex Purification and Identification by Mass Spectrometry, 01/10/2007, North Bethesda, MD, GTL: Genomics to Life Grantees' Workshop, [Biggin_2.pdf]This subproject of the Protein Complex Analysis Project (PCAP) is developing several complementary high throughput pipelines to purify protein complexes from D. vulgaris, identify their polypeptide constituents by mass spectrometry, and determine their stoichiometries. Our goal is to determine an optimum strategy that may include elements of each purification method. These methods will then be used as part of PCAP’s effort to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites. Our first purification approach is a novel “tagless” method that fractionates the water soluble protein contents of a bacterium into a large number of fractions, and then identifies the polypeptide composition of a rational sampling of 10,000 – 20,000 of these fractions using MALDI TOF/TOF mass spectrometry. Our second purification approach for water soluble proteins uses and extends the proven Tandem Affinity Purification method (TAP), in which tagged versions of gene products are expressed in vivo and then used to purify the tagged protein together with any other endogenous interacting components. Our third and fourth approaches are specialized variants of the tagless and TAP methods that are being designed to capture membrane protein complexes. A major part of our effort is the design and construction of automated instruments to speed the throughput of protein purification and sample preparation prior to mass spectrometry, and the development of rapid mass spectrometry data analysis algorithms. Once fully established, we will use our optimized methods to catalog as thoroughly as practicable the repertoire of stable heteromeric complexes in wild type cells grown under normal conditions, as well as identify a number of larger homomeric complexes. We will then examine changes in the composition of protein complexes in cells with perturbed stress response pathways. Response pathways will be perturbed either by growing cells in the presence of stressors, including nitrite, sodium chloride, and oxygen, or by mutating cells to delete a component of a stress response pathway. Purified heteromeric and homomeric complexes larger than 250 kDa are being provided to the EM Subproject to allow their structures to be determined and any stress induced changes in conformation to be detected. All of these data will be correlated by PCAP’s Bioinformatics Subproject with computational models of stress response pathways that are currently being established by the Environmental Stress Pathways Project (ESPP). Our results for the first year of the project are as follows. Tagless purification of water soluble complexes. We have developed an optimized four-step fractionation scheme for the tagless purification strategy and have used it to identify and purify 15 homomeric and heteromeric water soluble protein complexes from D. vulgaris. We have established an efficient, highly reproducible mass spectrometry sample preparation protocol that uses 96-well PVDF multiscreen plates, which will greatly aid high throughput analysis. This sample preparation method is effective for the iTRAQ methodology we have adopted to help quantitate the relative abundances of polypeptides in different chromatographic fractions. Methods for preparing protein samples suitable for single particle EM analysis are being refined, including the use of different crosslinking reagents to stabilize complexes on EM grids. To date, five complexes have been sent to the EM Subproject for structural determination. As a result a 17 Ĺ resolution structure of the 1 MDa Pyruvate Ferredoxin Oxidoreductase complex has been obtained.
Tagless purification of membrane complexes. We have developed an improved strategy to isolate membrane protein complexes that uses a multi-step membrane solubilization approach in which inner and outer membrane proteins are processed sequentially. The choice of detergent was shown to be critical, especially for the isolation of outer membrane protein complexes. Our methods for preparing samples for mass spectrometry analysis have been improved, particularly in the area of membrane protein native PAGE. Five membrane protein complexes have been identified and several others have been purified, ready for mass spectrometry analysis. Large-scale application of these methods is expected to facilitate the isolation and identification of substantially more complexes over the next project year. Tandem Affinity Purification of water soluble complexes. We have commenced trials of different TAP tag combinations for protein complex purification from D. vulgaris. Initial tests have compared the efficiency and observed non-specific binding properties of Sequential Peptide Affinity (SPA) tag, which is composed of Calmodulin Binding Peptide (CBP) and FLAG affinity purification tags, and the Strep-TEV-FLAG (STF) tag, which is similar to SPA but with CBP replaced by a Streptavidin tag. We have confirmed that both tags can purify proteins synthesized in D. vulgaris and are currently testing an expanded set of tagged proteins. Once completed, high-throughput methods currently being developed will be used to construct tagged D. vulgaris genes rapidly and efficiently. Automation of protein complex purification. We have developed a prototype multi-channel, native gel electrophoresis instrument for high resolution protein separation and automated band collection. This instrument can elute a protein band into a 200 µl fraction (about 60% of the band), without noticeable loss of sample. The use of this free-flow electrophoresis apparatus will greatly assist our efforts to achieve high throughput and provide an additional means of obtaining specimens in amounts appropriate for EM studies. Mass spectrometry. Optimization of MALDI TOF/TOF MS/MS conditions is necessary to maximize the quality and consequently the information content of the data. Resolution of the precursor selection window, number of laser shots, collision energy and collision gas pressure have been evaluated from the point of view of the success rate of protein identification and quality of the iTRAQ-based quantitation. Ultimately our high throughput mass spectrometry workflow will employ highly customized information-dependent selection of precursors for MS/MS. We have begun evaluation of different aspects of iterative MS/MS acquisition routines with the aim of limiting collection of redundant data on proteins already identified and focusing on reliable quantification and identification of less abundant species. The strategy employs collection of MS and very limited MS/MS during the first iteration followed by MS/MS acquisition performed in discrete stages, with each stage building upon a combination of results of current and preceding analyses of adjacent fractions within the same protein complex separation step. We have also are developing algorithms and graphical display tools for identifying protein complexes from mass spectrometry data, including a method for cluster analysis of tagless iTRAQ data to allow for detection of comigrating polypeptides and hence putative protein complexes. PresenterBiggin, Mark D. Funding SourceProtein Complex Analysis Project (PCAP) KeywordsProteomics |
| 186 |
Sapra, Rajat , Sandia National Laboratories Redox Proteomics In Desulfovibrio vulgaris Hildenborough: Search For Proteins That Mediate Stress Response via Post-Translational Modification of the Cys residues., 11/10/2006, Sudar Auditorium, Berkeley West Biocenter, VIMSS Monthly Meeting, [Modificomics_Sapra_11102006.ppt]  Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate reducing bacterium that grows in the absence of oxygen. From a physiological as well as ecological perspective, anaerobic bacteria have to overcome oxygen stress, the presence of which leads to the formation of reactive oxygen species (ROS). Anaerobic bacteria have evolved different mechanisms to overcome the stress induced by the presence of ROS. These include strategies to remove the ROS using post translational modification of the proteins, specifically the modifications of Cys residues in proteins, and to repair the damage in the cell to minimize the deleterious effects of the ROS associated stress. We are investigating the mechanisms by which DvH counters stress induced by oxygen as well as other redox stressors using a combination of 2D-DIGE and Isotope Coded Affinity Tag (ICAT) proteomics. I will discuss the challenges and progress that we have made towards identifying the proteins that are post-translationally modified in response to the redox stress.
PresenterSapra, Rajat Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsExtremophiles, Metabolomics, Stress Response, Sulfate Reducers |
| 187 |
Zhou, Jizhong Microbial Functional Genomics, Genomic Technologies and Environmental Applications, 11/07/2006, Los Alamos, NM, Invited talked, Genomcs Division, Los Alamos National Laboratorytbd PresenterZhou, Jizhong Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Functional Genomics |
| 188 |
Zhou, Jizhong Microbial Functional Genomics, Genomic Technologies and Environmental Applications, 10/27/2006, Clemson, SC, Invited talked, Department of Genetics and Biochemistry, Clemson Universitytbd PresenterZhou, Jizhong Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Functional Genomics |
| 189 |
M. W. Fields, T. Yan, X. Liu, C. E. Bagwell, S. L. Carroll, P. M. Jardine, D. B. Watson, C. S. Criddle, T. C. Hazen, and J. Zhou Identification of Different Relationships Between Contaminated Groundwater Samples Based Upon Extensive Geochemical Data or Multiple Gene Sequences from Microbial Communities, 10/24/2006, Oak Ridge, TN, Oak Ridge FRC Workshoptbd PresenterM. W. Fields Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioremediation, Evolutionary Biology, Extremophiles, Field Studies, Stress Response, Sulfate Reducers |
| 190 |
Hazen*, T. C., B. Faybishenko, E. Brodie, D. Joyner, S. Borglin, R. Chakraborty, M. Conrad, T. K. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett, and S. Koenigsberg. Invited. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor., 10/23/2006, Oak Ridge, TN., DOE ERSP annual field workshop., [hazenHanford100H.pdf]To carry out field investigations to assess the potential for immobilizing Cr(VI) in groundwater using lactate-stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site, and to determine critical community structure changes and stressors that would enable control and predictions of fundamental biogeochemistry that enables this bioremediation strategy for Cr(VI). PresenterHazen, T. C. Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBioremediation, Environmental Genomics, Extremophiles, Field Studies, Stress Response, Sulfate Reducers |
| 191 |
He, Zhili, Terry J. Gentry, Liyou Wu, Christopher W. Schadt, Joy D. Van Nostrand, Weimin Wu, Jost Liebich, Song C. Chong, Baohua Gu, Phil Jardine, Craig Criddle, David Watson, and Jizhong Zhou Monitoring of in situ Bioremediation Efficiency at the FRC Sites Is Revealed by a Comprehensive Functional Gene Array , 10/23/2006, Oak Ridge, TN, 2006 Fall ERSP Meeting, [Oct06-ZhiliHe_Monitoring.pdf]A novel comprehensive functional gene microarray, termed GeoChip, has been developed. This array contains 24,243 oligonucleotide (50mer) 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. The developed GeoChip was used to track the dynamics of metal-reducing bacteria and associated communities for an in situ bioremediation project at the FRC site in Oak Ridge. This is the first demonstration that uranium concentrations can be reduced to below the USA EPA maximum contaminant level (MCL) for drinking water. Genes known to be involved in U reduction were detected in the FRC bacterial community. This is the first comprehensive microarray available for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions. PresenterHe, Zhili Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioremediation, Microarrays, Stress Response |
| 192 |
Hemme, Christopher L., Terry J. Gentry, Ye Deng, Zhili He, Liyou Wu, Matthew W. Fields, Chris Detter, Kerrie Barry, David Watson, Paul Richardson, Terry Hazen, James Tiedje, Eddy Rubin and Jizhong Zhou Adaptation of a Microbial Community to Stress Conditions Prevalent at the FRC., 10/23/2006, Oak Ridge, TN, 2006 Fall ERSP Meeting, [Oct06-ZhiliHe_Adaptation.pdf]The majority of microorganisms in nature are unculturable by traditional methods and thus little is known about their genetic properties, biochemical functions, and metabolic characteristics. Although sequence determination of the microbial community ‘genome’ is now possible with high throughput sequencing technology, the complexity and magnitude of most microbial communities make meaningful data acquisition and interpretation difficult. Therefore, we are sequencing groundwater microbial communities with manageable diversity and complexity (~10-400 phylotypes) from the U.S Department of Energy’s Environmental Remediation Sciences Program (ERSP) Field Research Center (FRC), Oak Ridge, TN. The microbial community has been sequenced from a groundwater sample contaminated with very high levels of nitrate, uranium and other heavy metals and pH ~3.7. Three clone libraries with different DNA fragment sizes (3, 8 and 40 kb) were constructed, and 50-60 Mb raw sequences were obtained using a shotgun sequencing approach. The raw sequences were assembled into 421 major contigs totaling ~8 Mb which were further assembled into 224 scaffolds (1.8 kb-2.4 Mb). Preliminary binning of the scaffolds suggest 5 primary groupings (2 Frateuria-like γ-proteobacteria, 1 Burkholderia-like β-proteobacteria, 1 Herbaspirillum-like β-proteobacteria and 1 Afipia-like a-proteobacteria). Metabolic network reconstruction suggest rapid and specific adaptations to the geochemical conditions prevalent at the site. Furthermore, the low level of strain diversity observed across the entire metagenome and the random distribution of SNP’s suggest a recent population bottleneck. Further analysis of the metagenome suggests lateral gene transfer of contaminant resistance genes between community members. We hypothesize that the major adaptive response within the community following site contamination resulted from lateral gene transfer events between the surviving community members followed by adaptive evolution of individual genetic elements. These adaptive events would in turn be expected to trigger selective sweeps which would further reduce diversity within the community. PresenterHe, Zhili Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioremediation, Microarrays, Stress Response |
| 193 |
Hazen, T. C. Invited. October 2006, Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution., 10/07/2006, Logan, UT, Utah State Universitytbd PresenterHazen, T. C. Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Field Studies, Functional Genomics, Metabolomics, Metagenomics, Proteomics, Transcriptomics |
| 194 |
Zhou, Jizhong Microbial Functional Genomics, Genomic Technologies and Environmental Applications, 09/29/2006, Berkeley, CA, Lawrence Berkeley National Laboratorytbd PresenterZhou, Jizhong Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Functional Genomics |
| 195 |
Zhou, Jizhong (Joe) Genomics of Ethanol Producing Bacteria, 09/28/2006, Lake Arrowhead, CA, 14th International Conference on Microbial Genomestbd PresenterZhou, Jizhong (Joe) Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsComparative Genomics, Environmental Genomics, Functional Genomics, Transcriptomics |
| 196 |
Zhou, Jizhong Development and Application of Functional Gene Arrays for Assessing Microbial Community Structure and Functions in Natural Settings, 09/28/2006, University of California, Berkeley, CA, Department of Environmental Sciences, Policy and Managementtbd PresenterZhou, Jizhong Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Field Studies, Functional Genomics, Microarrays |
| 197 |
Dehal, Paramvir S. Handling large metagenomics data bases: requirements for an oil sands project, 09/27/2006, Calgary, Alberta, Canada, Genome Alberta Workshoptbd PresenterDehal, Paramvir S. Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioremediation |
| 198 |
Yinjie J. Tang, Adam L. Meadows, Jeannie Chu, Jay D. Keasling Flux analysis of anaerobic carbon metabolic pathways in Shewanella oneidensis MR-1 using isotopic metabolite labeling , 09/11/2006, San Francisco, CA, American Chemical Society 232nd National Meeting & Exposition, [Anaerobic_pathways_Tang2006.pdf] It 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 indicates 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 glyoxlate synthesized from 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 anaplerotic (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 condition, i.e., TMAO reducing condition. A flux distribution data according to our proposed pathway is estimated for both TMAO and fumarate reduction conditions.
PresenterYinjie J. Tang Funding SourceEnvironmental Stress Pathway Project (ESPP), Metabolomics KeywordsMetabolomics |
| 199 |
Zhou, Jizhong Functional Gene Arrays: Current Status, Challenges and Future, 09/11/2006, Okazaki, Japan, The 4th Okazaki Biology Conferencetbd PresenterZhou, Jizhong Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsFunctional Genomics, Microarrays |
| 200 |
Tang, Yinjie Pathway confirmation and flux analysis using 13C isotopic labeling of metabolites in Desulfovibrio vulgaris Hildenborough via mass spectrometry, 09/08/2006, LBNL Sudar Auditorium; Berkeley, CA, VIMSS Monthly Meeting, [vimss_090806_Tang.ppt] The metabolic pathways and flux distributions of the environmentally
important, sulfur reducing bacterium, Desulfovibrio vulgaris
Hildenborough were determined using 13C tracer experiments and Fourier
Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry. The flux
distribution results indicate the lack of an oxidatively functional TCA
cycle and an incomplete pentose phosphate pathway, both of which are
supported by the genome annotation and enzyme activity assays. The data
also indicate that 5,10-methyl-THF production occurs via serine
degradation. Although the genome annotation implicates a
ferredoxin-dependent oxoglutarate synthase homolog in the TCA cycle, no
evidence supports the use of this reaction. High resolution FT-ICR MS
measurements confirmed the presence of an atypical citrate synthase (the
citrate produced is the isotopic antipode of the citrate found in most
microorganisms) by locating the carbon position in two key amino acids
(aspartate and glutamate). These findings enable a better understanding
of the relation between the genome annotation and actual metabolic
pathways of D. vulgaris, and demonstrate FT-ICR MS as a powerful tool
for flux analysis, overcoming problems in both GC-MS and NMR. PresenterTang, Yinjie Funding SourceEnvironmental Stress Pathway Project (ESPP), Metabolomics, Protein Complex Analysis Project (PCAP) KeywordsFunctional Genomics, Metabolomics, Sequencing, Sulfate Reducers |
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