Systems Biology for DOE Energy and Environmental Missions Systems Environmental Microbiology The Virtual Institute of Microbial Stress and many unique aspects of studying such complex systems. For Survival VIMSS:ESPP example, the study of the D. vulgaris / Methanococcus mari- paludis syntrophic co-culture (serving as a model of naturally occurring SRB/ Methanogen interactions) required optimi- GTL zation of microarray hybridization methods and an alternate workflow for iTRAQ proteomics application. Our team also has advanced tools for metabolite level analysis, such as a 13C isotopomer based flux analysis which provides valuable ESPP Functional Genomics and Imaging information about bacterial physiology. However the study Core (FGIC): Cell Wide Analysis of of individual organisms in a mixed culture using existing Metal-Reducing Bacteria flux analysis methods is difficult since the method typically relies on amino acids from hydrolyzed proteins from a Aindrila Mukhopadhyay,1,6* Edward Baidoo,1,6 Kelly homogenous biomass. To overcome the need to separate the Bender5,6 ([email protected]), Peter Benke,1,6 target organism in a mixed culture, we successfully explored Swapnil Chhabra,1,6 Elliot Drury,3,6 Masood Hadi,2,6 Zhili the idea that a single highly-expressed protein could be used He,4,6 Jay Keasling1,6 ([email protected]), Kimberly to analyze the isotopomer distribution of amino acids from Keller,3,6 Eric Luning,1,6 Francesco Pingitore,1,6 Alyssa one organism. An overview of there studies and key obser- vations are presented. Redding,1,6 Jarrod Robertson,3,6 Rajat Sapra,2,6 Anup Singh2,6 ([email protected]), Judy Wall3,6 (wallj@ Additionally we continued to collect cell wide data in missouri.edu), Grant Zane,3,6 Aifen Zhou,4,6 and Jizhong Shewanella oneidensis and Geobacter metallireducens for Zhou4,6 ([email protected]) comparative studies. Improved methods for extraction and high throughput of metabolite analysis using CE-MS and 1 Lawrence Berkeley National Laboratory, Berkeley, LC-MS were applied to several studies underway in ESPP, 2 Calif.; Sandia National Laboratories, Livermore, Calif.; many of which are also required quantitation. In collabora- 3 4 University of Missouri, Columbia, Mo.; University of tion with Computational Core, a novel FTICR-MS based 5 Oklahoma, Norman, Okla.; Southern Illinois University, method for a comparative 12C/13C based metabolite analysis 6 Carbondale, Ill.; and Virtual Institute of Stress and was also developed to enable a direct comparison of control Survival (vimss.lbl.gov). and experimental samples for relative quantitation. Project Goals: The primary goal of the Environmental Improved methods for generating stable knockout mutants Stress Pathway Project (ESPP) is a rigorous understand- and marker-less mutants in D. vulgaris has been now been ing of the sulfate reducing bacterium (SRB), Desulfovibrio widely used to follow up hypothesis from a majority of our vulgaris Hildenborough physiology and its ability to stress response and most valuably in confirming candidates survive in its environment. for “missing steps” in D. vulgaris metabolism. Sets of tar- geted mutants are also being constructed to study the large The primary goal of the Environmental Stress Pathway number of two component signaling systems in D. vulgaris. Project (ESPP) is a rigorous understanding of the sulfate To ensure a complete understanding of regulatory mecha- reducing bacterium (SRB), Desulfovibrio vulgaris Hildenbor- nisms, the study alternative regulatory mechanisms such as ough physiology and its ability to survive in its environment. small non-coding RNAs are also underway. In collaboration This knowledge provides the basis for discerning the bio- with the computational core, work is in progress to set up geochemistry at metal contaminated sites, for bioremedia- searchable databases of all our large data sets, including tion and natural attenuation for toxic metals. The FGIC proteomics, metabolite and flux data. focuses on mapping these responses at a cell wide level using systems biology approaches. In the last one year, our meth- Acknowledgements ods that have been optimized and utilized over the years to This work was part of the Virtual Institute for Microbial Stress study a variety of growth/ stress conditions and mutants, and Survival (http://VIMSS.lbl.gov) supported by the U. S. were extended to study more environmentally relevant Department of Energy, Office of Science, Office of Biological physiological conditions and the interaction of D. vulgaris and Environmental Research, Genomics:GTL program through contract DE-AC02-05CH11231 between Lawrence Berkeley with other microbes. National Laboratory and the U. S. Department of Energy. Generating high quality biomass continues to be a critical aspect for all our functional genomics studies. This was found to be especially important for complex conditions such as biofilm formation, long term exposure to stress and the study of mixed cultures containing multiple organ- isms. These studies were conducted in close collaboration with the Applied and Environmental Core and additional methods development had to be undertaken to address 86 * Presenting author Systems Environmental Microbiology GTL these data implicate that Dv-sRNA2 and DVU0678 are involved in the D. vulgaris heat shock response, studies involving other stressors are currently underway. New strate- Analysis of a Desulfovibrio vulgaris Small RNA gies for constructing over-expression strains are also being and Its Target Under Various Stress Conditions developed to help predict the role individual sRNAs have on the physiology and transcriptional response of D. vulgaris Andrew S. Burns1,2 and Kelly S. Bender1,2* (bender@ under multiple environmental conditions. micro.siu.edu) Acknowledgements 1Microbiology Dept., Southern Illinois University, This work was part of the Virtual Institute for Microbial Stress 2 and Survival (http://VIMSS.lbl.gov) supported by the U. S. Carbondale, Ill.; and VIMSS (Virtual Institute for Department of Energy, Office of Science, Office of Biological Microbial Stress and Survival), http://vimss.lbl.gov and Environmental Research, Genomics:GTL program through contract DE-AC02-05CH11231 between Lawrence Berkeley Project Goals: Our goal is to understand the intricate National Laboratory and the U. S. Department of Energy. regulatory cascades involved in how the model metal reducer Desulfovibrio vulgaris and its relatives respond to stressors in contaminated sites. GTL One of the aims of the Environmental Stress Pathway Proj- ect is to elucidate regulatory networks critical to processes Functional Characterization of Microbial of interest to the DOE. As such, our goal is to understand the intricate regulatory cascades involved in how the model Genomes by Tagged Transposon Mutagenesis metal reducer Desulfovibrio vulgaris and its relatives respond Adam Deutschbauer1,4* ([email protected]), to stressors in contaminated sites. One approach we are tak- Julia Oh,2 Morgan Price,1,4 Jennifer Kuehl,1,4 Paramvir ing to meet this challenge is the identification and analysis Dehal,1,4 Corey Nislow,3 Ronald W. Davis,2 and Adam P. of small non-coding RNA molecules (sRNAs). Ranging in 1,4 size from 20-200 nucleotides, sRNAs predominantly affect Arkin gene regulation by binding to complementary mRNA in an 1Lawrence Berkeley National Laboratory, Berkeley, anti-sense fashion and therefore provide an immediate regu- 2 latory response independent of protein modification. Here Calif.; Stanford Genome Technology Center, Stanford, Calif.; 3University of Toronto, Toronto, Ontario, Canada; we report the analysis of Dv-sRNA2, a molecule previously 4 identified from a random small RNA clone library, and its and VIMSS (Virtual Institute for Microbial Stress and target gene DVU0678- a hypothetical protein only present Survival) http://vimss.lbl.gov in the D. vulgaris strains Hildenborough and DP4. Project Goals: A primary goal of the Environmental Stress Pathway Project (ESPP) is a systems-level model While expression of Dv-sRNA2 has been confirmed, of sulfate-reducing bacteria (SRB) metabolism, stress its regulatory role is currently unknown. Dv-sRNA2 is responses, and gene regulation. However, current sys- located in the same chromosomal region as its putative tems-level analyses of less studied bacteria such as SRBs target DVU0678, but on the opposite strand. As such, a are limited by the presence of numerous uncharacterized Dv-sRNA2 deletion mutant cannot be constructed without genes and an over reliance on annotations from well stud- affecting the expression of DVU0678. To circumvent ied bacteria such as E. coli. this problem, the gene encoding Dv-sRNA2 was cloned into the stable vector pMO719 to elucidate the effects A primary goal of the Environmental Stress Pathway of over-expression. This construct resulted in a strain Project (ESPP) is a systems-level model of sulfate-reducing (KB100) containing two copies of Dv-sRNA2 under the bacteria (SRB) metabolism, stress responses, and gene control of their native promoter. Phenotypic analysis of regulation. However, current systems-level analyses of less the KB100 strain compared to a control strain harboring studied bacteria such as SRBs are limited by the presence an empty vector indicated no difference in growth under of numerous uncharacterized genes and an over reliance normal 37ºC/pH 7 and pH 6 growth conditions. However, on annotations from well studied bacteria such as E. coli. a slight increase in growth rate was observed