Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Lawrence Berkeley National Laboratory
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The Virtual Institute of Microbial Stress and Survival (VIMSS): Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Microbes
Carl Abulencia4, Eric Alm1, Gary Andersen1, Adam Arkin1* (APArkin@lbl.gov), Kelly Bender5, Sharon Borglin1, Eoin Brodie1, Swapnil Chhabra3, Steve van Dien6, Inna Dubchak1, Matthew Fields7, Sara Gaucher3, Jil Geller1, Masood Hadi3, Terry Hazen1, Qiang He2, Zhili He2, Hoi-Ying Holman1, Katherine Huang1, Rick Huang1, Janet Jacobsen1, Dominique Joyner1, Jay Keasling1, Keith Keller1, Martin Keller4, Aindrila Mukhopadhyay1, Morgan Price1, Joseph A. Ringbauer, Jr.5, Anup Singh3, David Stahl6, Sergey Stolyar6, Jun Sun4, Dorothea Thompson2, Christopher Walker6, Judy Wall5, Jing Wei4, Denise Wolf1, Denise Wyborski4, Huei-che Yen5, Grant Zane5, Jizhong Zhou2, and Beto Zuniga6
1Lawrence Berkeley National Laboratory, Berkeley, CA; 2Oak Ridge National Laboratory, Oak Ridge, TN; 3Sandia National Laboratories, Livermore, CA; 4Diversa, Inc., San Diego, CA; 5University of Missouri, Columbia, MO; 6University of Washington, Seattle, WA; and 7Miami University, Oxford, OH
Introduction
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.
Accomplishments of the Applied Environmental Microbiology Core (AEMC)
Characterization of the Environment. The AEMC has collected or completed basic analysis of the stressors present at a number of NABIR FRC site, and characterized the microbial community before and after stimulation using 16SRNA microarrays. Large insert cloning was used to characterize the enrichment of genomic functions in these environments. Diversity analysis of library clones revealed genes used in transport, small molecule binding, toxicity response and DNA synthesis, among others. We are now targeting primers for enrichment of signal transduction pathway components. In addition, nine D. vulgaris-like bacteria (DP1-9) were isolated from a metal impacted field site (Lake DePue, Illinois). All had identical 16S rRNA and dsrAB genes that were virtually identical to the orthologous genes of DvH. Complementary whole-genome microarray hybridization revealed that approximately 300 deleted genes were distributed in six regions of the chromosome, annotated as conserved/ hypothetical or phage related genes in DvH. We are now following up characterization of these phageless strains.
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. Response to oxygen stress, salt stress (shock and adaptation) and nitrate have been fully characterized in this way. In related work, we are developing laboratory systems that simulate environmental conditions than can not be achieved in pure culture, initially focusing on co-cultures of two different Desulfovibrio species (DvH and Desulfovibrio sp. PT2) syntrophically coupled to a hydrogenotrophic methanogen (Methanococcus maripaludis). Transcriptional dynamics of the co-culture has been measured by the FGC. In addition, a metabolic stoichiometric model has been constructed using flux balance analysis (FBA) to complement and direct experimental studies on the physiology of DvH growing either alone or in co-culture.
Accomplishments of the Functional Genomics Core
Genetics: To improve the genetic accessibility of DvH, we found the cells to be sensitive to the antibiotic Geneticin or G418, therefore, allowing kanamycin resistance to be used as a genetic marker. Using the modified mini-Tn5 from Bill Metcalf, we have been able to generate a library of transposon mutants that appear to be randomly inserted throughout the genome. Several putative regulatory genes were among those mutated and we are screening for mutants of specific phenotypes. We have generated tagged hspC and rpoB genes in single copy controlled by their native promoters to use for development of assays for protein complexes. We have established a procedure for making gene deletions in non-essential genes that introduces a unique oligonucleotide that can be used for mutant identification. With this procedure, we have generated a putative fur deletion that is increased four fold over the wild type in its resistance to manganese. We are also generating a library of histidine kinase (HK) knockouts. DvH has 69 HKs that govern signal transduction. A suicide vector has been designed and created to enable gene deletion and concurrent “bar-coding” of the chromosome. Our preliminary results include 6 potential knock-out mutants.
Transcriptomics: We have, to date, characterized five stresses in DvH and five 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.
Proteomics: We have developed three complementary proteomics methods to characterize protein expression in our microbes Differential In Gel Electrophoresis followed by MALDI-TOF and nanLC-ISI-QTOF, Isotope coded affinity tagging with tandem LC mass spec, and direct MS-MS. In addition, to characterize protein complexes we have developed both a high throughput cloning & expression of DvH proteins in E. coli and methods for expression of genetically-modified proteins at their native levels in the host organism. These proteins are then used as bait proteins to enable “pull-down” of associated proteins.
Metabolomics: We have set up and 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.
Accomplishments of the Computational Core
During the past year the computational core has focused on building the comparative and functional genomic analysis tools to aid in the prediction of regulatory networks in microbes, elucidate their evolutionary relationships and extract the most meaning from the functional genomics and phenotypic data described in the last two sections. We have developed an increasingly sophisticated experimental and data management system that centralizes and serves all VIMSS data and tracks the progress through experimental runs of the pipeline. One of the key technologies we have developed is a set of web-accessible comparative genomic tools (http://vimss.org) designed to facilitate multi-species comparison among prokaryotes. Highlights of the system accessible through the VIMSS website include operon and regulon predictions based on novel methods we have proven to work on a wide diversity of micro-organisms, a multi-species genome browser, a multi-species Gene Ontology browser, a comparative KEGG metabolic pathway viewer and the VIMSS Bioinformatics Workbench for in-depth sequence analysis. In addition, we provide an interface for genome annotation, which like all of the tools reported here, is freely available to the scientific community. This tool has been used successfully by a number of projects. In particular, an Joint Genome Institute Annotation Jamboree we ran to annotate D. desulfuricans G20 which will likely be reclassified as D. alaskensis. We have also been working on tools for modeling pathways and understanding how the molecular strategies we measure in the lab confer the ability to survive in the environment.
* Presenting author
