# Presentation: 482182

## ESPP: Environmental Stress Pathway ProjectAdam Arkin1,2, Terry Hazen2, Directors Inna Dubchak1, Matthew Fields4, Hoi-Ying Holman1, Jay Keasling1,2, Anup Singh8, David Stahl3, Judy Wall6, Jizhong Zhou5, Martin Keller71 Lawrence Berkeley National Lab, 2 U.C.-Berkeley, 3 . U. of Washington, 4 Miami U., 5 U. of Okalahoma , 6 U. of Missouri, Columbia, 7 Diversa Inc., 8 Sandia National Lab

- The mission of the Virtual Institute of Microbial Stress and Survival is to understand the molecular basis for the survival and growth of microbes in the environment. Towards this end VIMSS has designed a series of key protocols, experimental pipelines and computational analysis to support and coordinate research in this area. Our flagship project aims to elucidate the pathways and community interactions which underlie the ability of *Desulfovibrio vulgaris *Hildenborough (DvH) to survive in diverse, possibly contaminated environments and reduce metals. Their ability to reduce toxic Uranium and Chromium, major contaminants of industrial and DOE waste sites, to a less soluble form has made them attractive from the perspective of bioremediation.
- We are discovering the molecular basis for the physiology of these organisms first through characterization of the biogeochemical environment in which these microbes live and how different features of these environments affect their growth and reductive potential. We have created an integrated program through the creation of an experimental pipeline for the physiological and functional genomic characterization of microbes under diverse perturbations. This pipeline produced controlled biomass for a plethora of analyses as described below and is managed through workflow tools and a data management and analysis system. The effort is broken into three interacting core activities: The Applied Environmental Microbiology Core; the Functional Genomics Core; and the Computational Core. While the individual accomplishments of these cores may be founding more detail we summarize the highlights here.

**Characterization of the Environment.** The AEMC has been monitoring natural and stimulated  groundwater and soil communities in DOE NABIR Field Research sites. These sites are contaminated with different combinations of heavy metals, low pH, chlorinated solvents, nitrates and other cellular stressors. Strong correlations between the population growth of certain strains of bacteria including DvH like strains and metal reduction dynamics were observed.* D. vulgaris *strains from multiple sites have been enriched and isolated and some are undergoing sequencing now. A novel cloning method was used to amplify DNA and whole genomes from five separated contaminated sites to compare and contrast functional diversity among these sites and relate this diversity to the stability and metal-reduction efficacy of the communities at these sites (including *D. vulgaris)*. An initial survey of the signal transduction genes is underway.
**Biomass production and Characterization: **In the core pipeline experiments each microbe is first characterized physiologically using Omnilog phenotypic microarrays. A stressor condition is then applied to a large set of batch cultures and samples are collected periodically to obtain a time-series of cellular response. Each time-point is split so that the cells can be imaged, analyzed through synchrotron IR microscopy to measure the bulk physiological changes of the cells during their response, and determine the optimal time points to send to the functional genomics core (FGC) for transcript, protein and metabolite analysis. Twenty-three conditions in DvH and *Shewanella oneidensis* (So) have been produced including mutant studies and co-culture of DvH syntrophically coupled to a hydrogenotrophic methanogen (*Methanococcus maripaludis*). Using publicly available genomic and physiological data, we developed a stoichiometric metabolic model that predicts that both hydrogen and formate could be used as electron carriers.  This model was able to predict metabolite accumulation of *D. vulgaris* in monoculture and in the syntrophic coculture at multiple stages in their dynamic growth cycle, but there were discrepancies between the experimental data and model predictions at individual time points which can be explained by interesting observed growth dynamics.

**Genetics: **Our bar-coded deletion project proceeds apace and a number of deletions have been phenotyped by omnilog array and through the VIMSS physiological pipeline. New affinity tags have been developed for pull-down and molecular complex studies both for this and a collaborating project. We have also expanded our transposon mutagenesis library and have begun to array them for sequencing and phenotyping. The most interesting of these will be submitted to pipeline studies.
**Transcriptomics:** We have, to date, characterized seventeen stresses, growth phase conditions, or mutant responses in *DvH* and six in *S. oneidensis* and results are integrated with the VIMSS MicrobesOnline Database. New regulons and their cis-regulatory sequences have been discovered along with new hypotheses of the pathways by which both organisms respond to these different stressors. A number of papers are in press, submitted or are in preparation around this topic. Compendium analysis for DvH is underway. *Geobacter* stressors are coming online and will be used for the three organism cross comparison.
**Proteomics: **As part of the proteomics mission we have developed and compared and contrasted MS/MS, ICAT MS, ITRAQ MS, and DIGE (Differential In-gel Electrophoresis) MALDI to profile the protein abundance and protein abundance changes in response to stressors. The year has been spent in quantifying reproducibility and accuracy of these results and comparing their predictions to that of microarray. There were significant differences among the methods and with the microarray data, however, these highlight the different sensitivities of the instruments and the long cell-cycle times of the microorganism. Recent, longer term experiments show better agreement between the microarray and proteomic data. We are now targeting our proteomics efforts to track the changes in the key sulfate reducer signature genes (see below) and their linked stress response pathway regulators to generate a more limited but precise measure of both abundance and redox changes.
**Metabolomics: **We have optimized both Capillary electrophoresis (CE) and Liquid chromatography (LC) coupled with Mass spectrometry (MS) methods for characterization of metabolites. Metabolite extraction protocols have been developed for *DvH*. A new Fourier transform ion cyclotron resonance mass spectrometer has recently come online which will allow a much wider survey of metabolites without the need for external standards. We have identified the key pathways and metabolites on which we will focus to understand the sulfate-reduction and stress metabolism of *DvH *and it contrast with *S. oneidensis* and *Geobacter.*

- The computational core has continued its core development of MicrobesOnline as the core framework for comparative functional microbial genomics and for the dissemination and visualization of VIMSS data. The microarray database is now fully integrated with the website and proteomic, metabolomic and molecular interaction data will shortly follow. A highly curated database of cis-regulatory motifs and their regulators in a variety of organisms from Mikhail Gelfand is undergoing integration with MicrobesOnline and a suite of cis-regulatory prediction tools are being added to the informatics tools available on the site. The team has used the site to provide nearly automated data analysis for the microarray experiments above and has been used to understand the key biological mechanisms in a number of responses including cold and heat shock, low and high pH, low and high oxygen, salt and osmotic shock and adaptation, nitrate and nitrite stress, chromium reducing conditions, growth in co-culture with a methanogen, iron limitation and mutation in the fur gene. A new method called OpWise has aided in this analysis and uses prior computational core work in accurate operon prediction to exploit genomic architecture to get better estimates of gene expression changes and the systematic and non-systematic errors underlying the measurements. We are now in the process of integrating the new proteomic and metabolomic data into the framework. We already have a new interface for analysis and visualization for the phenotype microarray data which we are now using for quality control and analysis of mutant data.
- Along with the core platform development and data analysis and interpretation, the computational core has used MicrobesOnline as a platform for more general discovery and annotation. It is being used as an annotation tool for a number of new genomes and environmental sequences. For example, in collaboration with another project we have identified and annotated a novel sulfate reducing organism isolated from a deep South African goldmine. In addition, a set of signature genes that define sulfate reducing bacteria and Archaea were inferred and validated against the gene expression database. A set of *DVH *centered comparative pathway analyses for the metabolic, global regulatory, dissimilatory nitrogen oxide pathways were accomplished with Mikhail Gelfand’s group. New theories for the formation, maintenance, tuning, and death of operon structure were put forward and a comprehensive study on the evolution of histidine kinases was completed. This study also discovered that certain organisms are more likely to generate new kinases by lineage specific expansion and other to acquire them through horizontal gene transfer.

- 1.        Alm, E.J., Huang, K.H., Price, M.N., Koche, R.P., Keller, K., Dubchak, I.L., and Arkin, A.P. (2005) The MicrobesOnline Website for Comparative Genomics. *Genome Research*, **15**(7):1015-22
- 2.        Bender, K., Yen, H.-C., and Wall, J.D., Analyzing the metabolic capabilities of *Desulfovibrio* species through genetic manipulation,  *Biotechnology and Genetic Engineering Reviews,* **23**, in press
- 3.        Chhabra, S.R., He, Q., Huang, K.H., Gaucher, S.P., Alm, E.J., He, Z., Hadi, M.Z., Hazen, T.C., Wall, J.D., Zhou, J., Arkin, A.P. and A.K. Singh, Global Analysis of Heat Shock Response in *Desulfovibrio vulgaris* Hildenborough. *J. Bacteriol.*, in press
- 4.        DeSantis, T.Z., Stone, C.E., Murray, S.R., Moberg, J.P., and Andersen, G.L. (2005) Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray. *FEMS Microbiol Lett.*, Apr 15;**245**(2):271-278
- 5.        Fields, M.W., Yan, T., Rhee, S.-K., Carroll, S.L., Jardine, P.M., Watson, D.B., Criddle, C.S. and Zhou, J.  (2005)  Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste.   *FEMS Microbiol. Ecol.*
-            **53**(3):417-428
- 6.        Gao, W., Liu, Y., Zhou, J.-Z., and Hongjun, P. (2005) Effects of a Strong Static Magnetic Field on Bacterium Shewanella oneidensis: An Assessment by Using Whole Genome Microarray. *Bioelectromagnetics*, **26**(7): 558-563
- 7.        Gentry, T., Schadt, C., and Zhou, J. (2005) Microarrays for environmental applications. *Microbial Ecology*, in press (Invited)
- 8.        Gentry, T., Schadt, C., and Zhou, J. (2005) Microarrays for bacterial detection. In *Advanced Techniques for Diagnostic Microbiology* (Invited)
- 9.        Hazen, T. C. and Tabak, H.H.  (2005) Developments in Bioremediation of Soils and Sediments Polluted with Metals and Radionuclides: 2. Field Research on Bioremediation of Metals and Radionuclides.  *Reviews in Environmental Science and Biotechnology*, **4**(3):157-183
- 10.    Hazen, T. C. et al. (contributing author) (2005) SERDP and ESTCP Expert Panel Workshop on Research and Development Needs for the Environmental Remediation Application of Molecular Biological Tools. *Department of Defense SERDP & ESTCP*,  **October 2005**:36-38
- 11.    He, Z., Wu, L., Li, X., Fields, M., and Zhou, J.-Z. (2005) Empirical establishment of oligonucleotide probe design criteria using perfect match and mismatch probes and artificial targets. *Appl. & Environ. Microbiol.*, **71**(7):3753-3760
- 12.    He, Z., Wu, L., Fields, M., and Zhou, J.-Z. (2005) Comparison of microarrays with different probe sizes for monitoring gene expression. *Appl. & Environ. Microbiol.*, **71**(9):5154-5162
- 13.    Koenigsberg, S. S., Hazen, T. C., and Peacock, A. D. (2005) Environmental Biotechnology: a Bioremediation Perspective.  *Remediation Journal*, **15**:2-25
- 14.    Li*, Xingyuan, He*, Zhili, and Zhou, Jizhong **(***co-first author) (2005) Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation. *Nucleic Acids Research****, *****33**(19):6114-6123
- 15.    Liu, Y., Gao, W., Wang, Y., Wu, L., Liu, X., Yan, T., Alm, E., Arkin, A., Thompson, D.K., Fields, M.W., and Zhou, J. (2005) Transcriptome Analysis of *Shewanella oneidensis* MR-1 in Response to Elevated Salt Conditions. *J Bacteriol*, **187**(7):2501-2507
- 16.    Nielsen, J.L., Schramm, A., Bernhard, A.E., van den Engh, G.J., and Stahl, D.A. (2005) Flow Cytometry-assisted Cloning of Specific Sequence Motifs from Complex 16S ribosomal RNA Gene Libraries. *Appl. Environ. Microbiol*., in press
- 17.    Price, M.N., Alm, E.J., and Arkin, A.P. (2005) Interruptions in gene expression drive highly expressed operons to the leading strand of DNA replication. *Nucleic Acids Research.* **33**(10):3224-34
- 18.    Price, M.N., Huang, K.H., Arkin, A.P., and Alm, E.J. (2005) Operon Formation is Driven by Co-Regulation and Not by Horizontal Gene Transfer. *Genome Research*, **15**(6):80-19
- 19.    Price, M.N., Huang, K.H., Alm, E.J., and Arkin, A.P. (2005) A Novel Method for Accurate Operon Predictions in All Sequenced Prokaryotes. *Nucleic Acids Research*, **33**(3):880-892
- 20.    Price, M.N., Alm, E.J., and Arkin, A.P., OpWise: Operons aid the identification of differentially expressed genes in bacterial microarray experiments. *BMC Bioinformatics*, in press
- 21.    Price, M.N., Alm, E.J., and Arkin, A.P., The Histidine Operon is Ancient. *J. Molecular Evolution*, in press
- 22.    Rodionov, D., Dubchak, I.L., Arkin, A.P., Alm, E.J., and Gelfand, M.S. (2005) Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks. *PLOS Comp. Bio.*, in press
- 23.    Schadt C.W. and Zhou, J.-Z. (2005) Advances in Microarrays for Soil Microbial Community Analyses. In *Soil Biology Volume 3: Nucleic Acids and Proteins in Soil* (Smalla K., ed), Springer-Verlag, In Press (Invited)
- 24.    Schadt, C. W., Liebich, J., Chong, S. C., Gentry, T. J., He, Z., Pan, H. and Zhou, J.-Z. (2005) Design and Use of Functional Gene Microarrays (FGAs) for the Characterization of Microbial Communities. In Savidge T, Pothulakis H (eds) *Methods in Microbiology V34: Microbial Imaging*,    pp331-368, Academic Press, London (Invited)
- 25.    Thompson, D. K. (2005) Cellular Response of Shewanella oneidensis to Strontium Stress. *Appl. Environ. Microbiol*., in press
- 26.    Wagner, M., Smidt, H., Loy, A., and Zhou, J. (2005) Unravelling Microbial Communities with DNA-Microarrays: Challenges and Future Directions. *Microb. Ecol.*, in press
- 27.    Wu, L., Liu, X., Schadt, C. W., and Zhou, J.-Z.  (2005) Microarray-based analysis of subpicogram quantities of microbial community DNA. *PNAS*, in press
- 28.    Zhou, J.-Z. (2005) _[Impact of microarray technology and bioinformatics on microbial ecology](http://www.kenes.com/isme10/program/session1.asp?SessionId=S17&SSessionDate=8/24/2004)_, Minireview for the 10th International Symposium on Microbial Ecology. *Microbial Ecology,* in press ***2004***
- 29.    Arkin, A., Ambrosiano, J., Babnigg, G., Frank, E., Geist, A., Giometti, C., Jacobsen, J., Samatova, N., Slater, N, and Taylor, R. (2004) Genomics: GTL Data Standards. *Technical Reference*, **1.2**
- 30.    Gao, H., Wang, Y., Liu, X., Yan, T., Wu, L., Alm, E., Arkin, A., Thompson, D.K., and Zhou, J. (2004) Global Transcriptome Analysis of the Heat Shock Response of *Shewanella oneidensis*. *J. Bacteriol*, **186**(22):7796-7803
- 31.    McAdams, H., Srinivasan, B., and Arkin, A. (2004) The Evolution of Genetic Regulatory Systems in Bacteria. *Nature Reviews*, **5**:169-178
- 32.    Rodionov, D.A., Dubchak, I., Arkin, A., Alm, E., and Gelfand, M.S. (2004) Reconstruction of Regulatory and Metabolic Pathways in Metal-reducing Delta-proteobacteria. *Genome* *Biology*, **5**(11):R90
- 33.    Tiquia, S.M., Chong, S.C., Fields, M.W., and Zhou, J. (2004) Oligonucleotide-based functional gene arrays for analysis of microbial communities in the environment, *In* Molecular Microbial Ecology Manual. G. A. Kowalchuk, Netherlands Institute of Ecology Centre for Terrestrial Ecology (NIOO-KNAW), Heteren, The Netherlands; F.J. de Bruijn, Laboratory of Plant-Microbe Interactions (ex LBMRPM), UMR INRA-CNRS, France; I. M. Head, School of Civil Engineering and Geosciences, University of Newcastle, UK; A.D. Akkermans, Dept. of Microbiology, University of Wageningen & Research Centre, The Netherlands; J. D. van Elsas, Microbial Ecology, Groningen University Biological Center, The Netherlands (Eds.). Kluwer Academic Press ***2003***
- 34.    DeSantis, T., Dubosarskiy, I., Murray, S., and Andersen, G. (2003) Comphrehensive Aligned Sequence Construction for Automated Design of Effective Probes (CASCADE-P) using 16S rDNA. *Bioinformatics*, **19**:1461-1468
- 35.    Alm, E. and Arkin, A. (2003) Biological Networks. *Current Opinion in Structural Biology*, **13:**193-202
- 36.    Wolf, D.M. and Arkin, A.P. (2003) Motifs, modules and games in bacteria. *Current Opinion in Microbiology*, **6**(2):125-134
**Publications in Submission:**
- 37.    Brown, S.D, Martin, M.,  Huang, K., Alm, E., Yang, Y., Arkin, A., Zhou, J.-Z., and D. K. Thompson, Induction of Iron Transport Genes in Strontium-Stressed *Shewanella oneidensis*: Possible Involvement of Siderophores in Heavy Metal Tolerance, *AEM *(Submitted 2005)
- 38.    Fields, M. W., Bagwell, C. E., Carroll, S. L., Yan, T., Liu, X., Watson, D. B.   Jardine, P. M., Criddle, C. S., Hazen, T. C., and Zhou, J., Gene sequences as indicators of bacterial community responses to mixed-waste contamination, *Environ. Sci. Technol.*  (Submitted 2005)
- 39.    Gao, H., Thompson, D.K., and Zhou, J.-Z., Global Transcriptome Analysis of the Cold Shock Response of *Shewanella oneidensis, J. Bacteriol* (Submitted 2005)
- 40.    He, Q. Huang, K., He, Z., Alm, E.J., Fields, M. W., Hazen, T.C., Arkin, A.P.,  Wall, J.D. and Zhou, J.-Z., Energetic Consequences of Nitrite Stress in *Desulfovibrio vulgaris* Hildenborough Inferred from Global Transcriptional Analysis. *J. Bacteriol* (Submitted 2005)
- 41.    Holman, H.-Y. N., Lin, Z., Comolli, L.R., Downing, K.H., Hazen,  T.C., and E. Wozei, Real-Time Molecular Observation of Adaptation to Oxygen Stress in Sulfate-Reducing Bacteria. *Science,* LBNL-54957 (Submitted 2005)
- 42.    Kurowski, J., Sundararaja, A., Klonowska, A., Yan, T.,, Klingman, D.M., Duley, M., Zhou, J., and Fields, M.W. Deletion of a multi-domain PAS protein causes pleiotropic effects in *Shewanella* *oneidensis* MR-1.  *J. Bacteriol.*  (Submitted 2005)
- 43.    Leaphart, A.B., Thompson, D.K., Huang, K., Alm,E., Wan, X., Arkin, A., Brown, S.D., Wu, L., Yan, T., Liu, X., and Zhou, J., Transcriptome Analysis of *Shewanella oneidensis *Gene Expression in Response to Acidic and Alkaline pH Stress. *J. Bacteriol* (Submitted 2005)
- 44.    Li, X., He, Z. and Zhou, J.-Z., Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation, *Nucl. Acids Res.* (Submitted 2005)
- 45.    Price, M.N., Arkin, A.P., and Alm, E.J., The Life-cycle of Operons and Evidence for Selection on Gene Expression Patterns. *PLoS Genetics *(Submitted 2005)
- 46.    Mukhopadhyay, A., He, Z., Yen, H.-C., Alm, E.J., He, Q., Huang, K., Baidoo, E.,E., Chen, W., Borglin, S.C.,. Redding, A., Holman, H.-Y., Sun, J., Joyner, Katz, N., D.C., Keller, M., Oeller, P., Zhou, J., Arkin, A.P., Hazen, T.C., Wall, J., Wei, J.,Yang, Z., and Keasling, J.D., Salt stress in *Desulfovibrio vulgaris* Hildenborough: An integrated genomics approach. *Genome Research*  (Submitted 2005)
- 47.    Walker, C.B., Stolyar, S.S., Pinel, N., Yen, H.C., He, Z., Zhou, J., Wall, J.D., and Stahl, D.A., Recovery of temperate *Desulfovibrio vulgaris* bacteriophage on a novel host strain. *Environmental Microbiology *(Submitted 2005)
- 48.    Nitrite stress on Dv will be submitted to *Mol Microbiol* (Submitted 2005)

- This work was part of the Virtual Institute for Microbial Stress and Survival supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics Program:GTL via contract DE-AC03-76SF00098 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy.

| Cellular Responses to Changing Conditions in Desulfovibrio vulgaris and Shewanella oneidensis |
| --- |
| Comparative Analysis of Bacterial Gene Expression in Response to Environmental Stress  |
| Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments  |
| Evaluation of stress responses in sulfate-reducing bacteria through genome analysis: identification of universal responses  |
| H2 O2Induced Stress Responses of Shewanella oneidensis MR-1 |
| Investigation of Protein-Protein Interactions in the Metal-Reducing Bacterium Desulfovibrio vulgaris  |
| Metagenomic Analysis of Microbial Communities in Uranium-Contaminated Groundwaters  |
| Nitrate stress response in Desulfovibrio vulgaris Hildenborough: Whole-Genome Transcriptomics and proteomics analyses  |
| VIMSS Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers  |
| VIMSS Computational Core: Comparative Analysis of Regulatory Systems in Environmental Microbes  |
| VIMSS Functional Genomics Core Research on Stress Response Pathways in Metal-Reducers  |
| Protein Complex Analysis Project (PCAP): Data Management and Bioinformatics Subproject |
| Protein Complex Analysis Project (PCAP): Imaging Multi-Protein Complexes by Electron Microscopy |
| Protein Complex Analysis Project (PCAP): Microbiology Subproject |
| Protein Complex Analysis Project (PCAP): Multi-Protein Complex Purification and Identification by Mass Spectrometry |
| Protein Complex Analysis Project (PCAP): Project Overview |
| New Technologies for Metabolomics |
| Application of a Novel Genomics Technology Platform |
| Anaerobic Respiration Gene Regulatory Networks in Shewanella oneidensis MR-1 |

- VIMSS Front Page
- http://vimss.lbl.gov

- MicrobesOnline
- http://microbesonline.org

- EIDR
- http://vimss.lbl.gov/EIDR

- BioFiles
- https://vimss.lbl.gov/perl/biofiles

- GreenGenes
- http://greengenes.lbl.gov

- The AEMC has a reliable, robust and repeatable procedure for production of extremophile biomass production under a variety of stressors.
- The AEMC has pioneered phenotype microarray procedures for extremophiles and anaerobes.
- The AEMC has pioneered the use of Synchrotron FTIR Microscopy to confrm stress response conditions and pick time points for biomass sampling under such stress.
- VIMSS created a flexible microbial physiological pipeline comprising biomass characterization, cellular imaging, transcriptomics, proteomics, and metabolomics, that can rapidly upgraded and repurposed for new projects.
- This pipeline lays the foundation for integration of functional genomic and physiological data and facilitates a synergetic approach to physiological characterization of microorganisms more efficiently than one lab could alone.
- VIMSS pipeline now being used for projects beyond ESPP such as the PCAP project.
- AEMC has identified Dv at DOE contaminated sites and has explored correlation of Desulfovibrio growth with metal reduction..
- In collaboration with NABIR projects on Uranium contamination, AEMC has begun to link natural stimulation studies to community measures of bacterial populations including Dv.
- AEMC has characterized contaminated environments to choose stressors for laboratory work.
- AEMC identified functional changes in bacterial systems under different stress conditions, nutrients, and niche, at FRC sites.
- AEMC has pioneered development of dual culture Dv and Mm in obligate syntrophic growth for studying this tiny community.
- AEMC has developed a flux-balance model for the syntrophic pair which explains many growth conditions and identifies the ech hydrogenase as dominant in proton/hydrogen pumping.
- The AEMC has isolated a number of subspecies of Dv including a phage deficient strain (DP4) that are now undergoing sequencing. They will also be tested phenotypically and in the co-culture to understand the variability of regulation in Dv.
- The AEMC has developed new techniques for metagenomics library construction and sequence from soil samples.
- The FGC has developed efficient transposon and well-defined knockout genetics for Dv that can be routinely used to discover genes important for fitness and to test predictions from the functional genomic data.
- The FGC has developed high-efficiency affinity tags for exogenous and endogenous pull-down experiments to find molecular complexes. These support both ESPP and PCAP.
- The FGC developed Differential In-Gel Electrophoresis for identification of post-translational and redox state of proteins.
- The FGC is able to compare and contrast three different industry standard proteomics techniques to each other and to functional genomic data for the first time. This allows new measures quality and consistency and the discovery of new biology.
- The CC has developed a community comparative microbial genomics/physiology database and workbench called MicrobesOnline that is now being used for annotation of new organisms, functional genomic analysis, and understanding microbial regulatory networks.
- The CC has developed a number of statistical analysis tools for improving and comparing results from the VIMSS pipeline.
- The CC has developed methods for predicting operons and regulons in uncharacterized organisms and has exploited these results to understand operon evolution, better analyze microarray data, and detect cis-regulatory sequences.
- The CC has used MicrobesOnline in collaboration with Michael Gelfand and his RegTransDB to infer global regulatory networks such as Fur and Per and metabolic pathways in Dv and across organisms. MicrobesOnline and RegTransDB have been linked for advanced cis-regulatory site prediction.
- The CC has discovered that delta proteobacteria like Dv and Geobacter have an abnormally high abundance of signaling components that arise due to linear specific expansion unline model organisms like B. subtilis and E. coli which seem to acquire new histidine kinases by HGT. The AEMC has targetted a number of the unique HPK families for enriching signalling molecules in the AEMC environmental libraries.
- The CC has  used comparative analysis across diverse sulfate reducers to discover a set of signature genes that are found in bacteria and Archaea with this phenotype. Coregulation of the signature genes was discovered in the VIMSS microarray compendium. A new thio-relay has been proposed based on this signature and the prediction is being followed up by deletion, protein complex studies, and by redox DIGE.
- Analysis of the VIMSS compendium and the Dv genome reveals that there is no generalized stress response with analogy to that found in *E. coli* or *B. subtilis*.  Instead, up-regulating of the inferred fur regulon seems to be a common motif in Dv stress response. The lack of a general stress response might represent the control the complex signaling system has of Dv physiology.
- The *fur, per, zur* regulons seem to have a unique pattern of regulation and there seems to be so siderophores for metal scavenging to be controlled by these regulatory.
- Dv demonstrates aunique nitrate response which may be important at the nitrate contaminated DOE sites. Full pipeline analysis of over ten stressors have demonstrated a stress response system in Dv very distrinct from the model organisms and sometimes demonstrates combinations of *E. coli-*like and *B. subtilis*-like responses.
- VIMSS now houses 4 new GTL projects, a NASA Astrobiology Project, a  DHS green genes project, ,and  2 NABIR projects.
- In an example from these collaborations, VIMSS has aided in the annotation of a metagenome from a deep subsurface gold mine. A nearly clonal population of a new speccies  of sulfate reducer was discovered with new classes transposons and a number of unique regulatory and gene features.