Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water. Cr(VI) is more soluble, toxic, carcinogenic, and mutagenic compared to its reduced form Cr(III). In order to stimulate microbially mediated reduction of Cr(VI), a polylactate compound HRC was injected into the chromium contaminated aquifers at site 100H at Hanford. Based on the results of the bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products, we recently investigated the diversity of the dominant anaerobic culturable microbial population present at this site and their role in Cr(VI) reduction. Positive enrichments set up at 30oC in the dark using specific defined anaerobic media resulted in the isolation of an iron reducing isolate strain HAF, a sulfate reducing isolate strain BLS and a nitrate reducing isolate, strain HLN. Preliminary 16S rDNA sequence analysis identifies strain HAF as Geobacter metallireducens, strain HLN as Pseudomonas stutzeri and strain BLS as a member of Geosinus species. Strain HAF isolated with acetate as the electron donor utilized propionate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Growth was optimal at 37oC, pH of 6.5 and 0% salinity. Strain HLN isolated with lactate as electron donor utilized acetate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Optimal growth was observed at 37oC, at a pH of 7.5 and 0.3% salinity. Anaerobic active washed cell suspension of strain HLN reduced almost 95f...lM Cr(VI) within 4 hours relative to controls. Further, with 100f...lM Cr(VI) as the sole electron acceptor, cells of strain HLN grew to cell nun1bers of 4.05x107/ml over a period of 24h after an initial lag, demonstrating direct enzymatic Cr(VI) reduction by this species. 10mM lactate served as the sole electron donor. These results demonstrate that Cr(VI) immobilization at the Hanford lOOH site could be mediated by direct microbial metabolism apart from indirect chemical reduction of Cr(VI) by end products of microbial activity.

The mechanisms of Desulfovibrio vulgaris Hildenborough responses to long-term NaCl exposure were studied by global transcriptional and metabolite analyses. The growth of D. vulgaris was inhibited by high salinity, and salt inhibition could be relieved by an addition of amino acids (e.g., glutamate, alanine) or yeast extract. Salt shock (sudden increase in salt concentration) and salt adaptation (inoculating cells in the medium containing high concentrations of salt) showed a significant difference in respective transcriptomes. Salt adaptation induced expression of genes involved in amino acid biosynthesis and transportation, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). Genes involved in energy metabolism, cell motility, and phage structures were repressed. Genes involved in Na+/H+ transport, K+ uptake and transportation, and proline biosynthesis and transportation were not significantly affected. Metabolite assays and external addition of amino acids into the growth medium of D. vulgaris suggest that amino acids, such as glutamate and alanine may accumulate as osmoprotectants in D. vulgaris. A conceptual model is proposed to link our observed results to currently available knowledge for further understanding the mechanisms of adaptation of D. vulgaris to sodium chloride.

Sulfate reducing bacteria (SRB), found widely in nature, use sulfate as the terminal electron acceptor in their respiratory cycle, leading to the production of hydrogen sulfide. These bacteria have both ecological and economic importance. SRB play a role in various biogeochemical cycles including the sulfur and carbon cycles. They have a negative economic impact on the oil industry, where their metabolism causes corrosion and clogging of machinery, and fouling of oil wells. However, they have also been shown to reduce and/or immobilize toxic water-soluble metals such as copper (II), chromium (IV) and uranium (VI), and thus are candidates for bioremediation applications. Desulfovibrio vulgaris Hildenborough (DvH) is a member of the most well studied genus of SRBs. A goal of the Environmental Stress Pathway Project (ESPP) in the Virtual Institute for Microbial Stress and Survival (VIMSS) is to understand the regulatory networks in DvH for applications to bioremediation. One aspect of this is the elucidation of protein post-translational modifications (PTMs) in DvH.PTMs play various roles in the cell. Some modifications play a role in protein structure, such as lipid anchors or some disulfide bonds. Others are directly involved in regulation of protein function such as phosphorylation and glycosylation. Still others arise through cellular damage such as irreversible oxidation events. Whatever the role these PTMs play, they must be characterized at the protein level because they are not directly coded for in the genome. Furthermore, DvH may be particularly likely to use PTMs as a regulatory mechanism: Evidence for this includes the observation that the DvH genome encodes an abnormal number of histidine kinases. Our goal is to determine the types of protein modifications that arise in DvH and how these modifications affect the ability of DvH to survive or adapt to its environment. This work leverages the unique resources of the Virtual Institute for Microbial Stress and Survival: Quality controlled biomass produced at LBL (Hazen lab) is used for all proteomic LC/MS/MS measurements at LBL (Keasling lab). Our initial survey of PTMs in DvH was obtained by mining these numerous proteomic LC/MS/MS data sets acquired over the course of ESPP for evidence of modified peptides. Data mining for PTMs is performed at Sandia National Labs. The searched-for modifications were determined based on literature precedence and a genome search for the existence of relevant transferases. To date we have found preliminary evidence for cysteine oxidation, lysine acetylation, and methylation of lysine and arginine. Data mining for additional PTMs is ongoing. Future work will focus on validation of these findings and determining which, if any, of these modifications play a regulatory role in DvH. Validation will require selective isolation of the proteins of interest for further characterization. Here, protein isolation is made possible through the work being performed at LBL and the University of Missouri to generate DvH mutants containing tagged versions of DvH proteins.