Construction of a tatA Desulfovibrio vulgaris Hildenborough

Abstract only availabletatA Desulfovibrio vulgaris Hildenborough is a member of the obligately anaerobic bacteria growing by sulfate respiration and involved in environmental biocorrosion of ferrous metals. It also shows potential for bioremediation of toxic metals. Because these important metabolic activities of D. vulgaris are directly linked to electron flow, a better understanding of energy generation is needed. A model for augmenting respiratory energy production through hydrogen cycling has been proposed. This controversial model requires a periplasmic hydrogenase. The genome sequence of D. vulgaris reveals genes for four different periplasmic hydrogenases, the roles of which are currently unclear. There are two primary systems of transport of proteins such as hydrogenases to the periplasm or outer cell membrane. Both the Sec and Tat protein export systems translocate proteins across the cytoplasmic membrane. The Sec pathway exports short unfolded proteins, while the Tat system (Twin Arginine Translocation) translocates longer prefolded proteins. The latter generally contain redox cofactors and share a consensus motif (S/T)-R-R-x-F-L-K recognized for export. The Tat system is found in most prokaryotic plasma membranes. The Tat protein export system is encoded by four genes in E. coli, tatA, tatB, tatC, and tatE. However, only three of these genes, tatA, tatB, and tatC, have been putatively identified in D. vulgaris. Removal of one or more tat genes from E. coli causes deficiency in the transport of proteins by the Tat system. We propose to test the hydrogen cycling model for energy generation by creating a tatA deletion mutant in D. vulgaris that should block the production of all periplasmic hydrogenases. An examination of the deletion mutant should reveal the contribution of the hydrogen cycle to the energy economy of D. vulgaris.Life Sciences Undergraduate Research Opportunity Progra

Genetics and Molecular Biology of the Electron Flow for Sulfate Respiration in Desulfovibrio

Progress in the genetic manipulation of the Desulfovibrio strains has provided an opportunity to explore electron flow pathways during sulfate respiration. Most bacteria in this genus couple the oxidation of organic acids or ethanol with the reduction of sulfate, sulfite, or thiosulfate. Both fermentation of pyruvate in the absence of an alternative terminal electron acceptor, disproportionation of fumarate and growth on H2 with CO2 during sulfate reduction are exhibited by some strains. The ability to produce or consume H2 provides Desulfovibrio strains the capacity to participate as either partner in interspecies H2 transfer. Interestingly the mechanisms of energy conversion, pathways of electron flow and the parameters determining the pathways used remain to be elucidated. Recent application of molecular genetic tools for the exploration of the metabolism of Desulfovibrio vulgaris Hildenborough has provided several new datasets that might provide insights and constraints to the electron flow pathways. These datasets include (1) gene expression changes measured in microarrays for cells cultured with different electron donors and acceptors, (2) relative mRNA abundances for cells growing exponentially in defined medium with lactate as carbon source and electron donor plus sulfate as terminal electron acceptor, and (3) a random transposon mutant library selected on medium containing lactate plus sulfate supplemented with yeast extract. Studies of directed mutations eliminating apparent key components, the quinone-interacting membrane-bound oxidoreductase (Qmo) complex, the Type 1 tetraheme cytochrome c3 (Tp1-c3), or the Type 1 cytochrome c3:menaquinone oxidoreductase (Qrc) complex, suggest a greater flexibility in electron flow than previously considered. The new datasets revealed the absence of random transposons in the genes encoding an enzyme with homology to Coo membrane-bound hydrogenase. From this result, we infer that Coo hydrogenase plays an important role in D. vulgaris growth on lactate plus sulfate. These observations along with those reported previously have been combined in a model showing dual pathways of electrons from the oxidation of both lactate and pyruvate during sulfate respiration. Continuing genetic and biochemical analyses of key genes in Desulfovibrio strains will allow further clarification of a general model for sulfate respiration

Nitrogen fixation by photosynthetic bacteria : (Rhodospirilum rubrum, Rhodopseudomonas capsulata, glutamine ,nitrogenase)

Biological nitrogen fixation is not only essential for world nitrogen balance but it is also an alternative to expensive commercial fertilizer for crop production. To achieve the maximum utilization of this natural process, an understanding of the mechanism of N[subscript 2] reduction and its regulation is being sought. The photosynthetic bacteria, in particular members of the Rhodospirillaceae, are attractive organisms for genetic and biochemical analyses of nitrogen fixation. Characterization of mutants of these bacteria derepressed for synthesis of the nitrogenase complex in the presence of ammonium salts supports a critical role for glutamine and [lowercase alpha]-ketoglutarate in the regulation of synthesis. In addition, a mechanism exists for activity control by covalent modification of one of the protein components of the complex. The signal for modification and the extent to which this control mechanism occurs in other diazotrophs are under investigation.JUDY D. WALL, 322 Chemistry Building, University of Missouri, Columbia, Missouri

Applied and Environmental Microbiology Gordon Research Conference

The main objective of the Gordon Research Conference on Applied and Environmental Microbiology was to present and discuss new, fundamental research findings on microorganisms, their activities in the environment, their ecosystem-level effects, and their environmental or commercial applications. To accomplish this goal, knowledge of microbial diversity, interactions and population dynamics was required. The genomic basis of microbial processes, the cycling of naturally occurring and hazardous substances, and methodologies to assess the functional relationships of microorganisms in their habitats were essential for understanding the ecological consequences of microbial activities and the formulation of generalizing principles. In the last decade, molecular technology has revealed that microbial diversity is far more extensive than the limited view obtained from culturing procedures. Great advances in environmental microbiology have resulted from the development and application of molecular approaches to ecology and molecular evolution. A further surprise resulting from the application of these new tools is the blurring of the distinction between pathogenic traits versus those considered non-pathogenic. This year's conference addressed the issues of biodiversity, its development, and the impact of stress on gene selection and expression. In addition microbial metabolic versatility with toxins such as heavy metals, antibiotics, and organic pollutants were discussed. The nine session topics were (1) biodiversity and the bacterial species, (2) mechanisms of biodiversification, (3) biofilms in health and environment, (4) a genomic view of microbial response to stress, (5) microbial use of toxic metals, (6) microbial mineral formation and dissolution, (7) power and limitations of antimicrobials, (8) biodegradation of organic pollutants, and (9) astrobiology. The Conference had an international profile: the Conference Vice-Chair, Dr. Gerard Muyzer, was from The Nether lands, 10 of the 28 speakers and 2 of the 9 discussion leaders were from outside the USA. The program was composed of speakers and discussion leaders (selected through open peer discussion) from a wide range of universities, institutes, government laboratories, and industries. The Conference has traditionally drawn many non-U.S. conferees. In typical Gordon Conference style, the Conference sessions were informal, off the record, and open to all participants for the expression of their views. Meetings were held in the morning and evening. Afternoons were available for participation in small, informal discussion groups. The latest and often unpublished findings were emphasized. Following each evening session, conferees were able to gather in a central location where additional discussion occurred. Poster sessions facilitated informal discussion in the afternoon and evenings. The entire atmosphere of the Conference was designed to foster informal interaction. In fact, publication of a proceedings was not permitted by the Gordon Conferences in order to promote discussion of unpublished data. This type of meeting is a valuable means of disseminating information and ideas in a way that cannot be achieved through the usual channels of publication and presentations at large scientific meetings. The primary criteria for admittance to this Conference were scientific commitment and, implicitly, an interest in active and meaningful participation in the discussions

Identification of Molecular and Cellular Responses of Desulfovibrio vulgaris Biofilms under Culture Conditions Relevant to Field Conditions for Bioreduction of

Desulfovibrio vulgaris Hildenborough forms biofilms under stress and nutrient limitation conditions. Analysis of the transcriptional responses to acid exposure suggested that genes encoding arginine and polyamine biosynthesis were increased in expression. A literature search showed that polyamines had been suggested to stimulate biofilm formation in some bacteria. Therefore, biofilm formation by D. vulgaris was then examined. Two different assay methods were used to estimate the biofilm formation. First, the classical crystal violet stainable material attached to glass test tubes was measured. Second protein attached to the test tube sides and especially precipitated at the bottom was measured

Genes for Uranium Bioremediation in the Anaerobic Sulfate-Reducing Bacteria

Surprising results were obtained following an attempt to induce or derepress the machinery for U(VI) reduction by growing Desulfovibrio desulfuricans G20 in the presence of 1 mM uranyl acetate. G20 cells grown on lactate-sulfate medium amended with U(VI) reduced uranium at a slower rate than cells grown in the absence of this metal. When periplasmic extracts of these cells were prepared, Western analysis of the proteins revealed that the cytochrome c3 was absent. This observation has been further investigated

Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome c3

The project, ''Reduction of U(VI) and toxic metals by Desulfovibrio cytochrome c3'', is designed to obtain spectroscopic information for or against a functional interaction of cytochrome c3 and uranium in the whole cells. That is, is the cytochrome c3 the uranium reductase? Our approach has been to start with purified cytochrome and determine any unique spectral disturbances during electron flow to U(VI). Then we will attempt to identify these signals emanating from cells actively reducing uranium. This project is being carried out in collaboration with Dr. William Woodruff at the Los Alamos National Laboratory where the spectral experiments are being carried out

Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome C3

The central objective of our proposed research was twofold: 1) to investigate the structure-function relationship of Desulfovibrio desulfuricans (now Desulfovibrio alaskensis G20) cytochrome c3 with uranium and 2) to elucidate the mechanism for uranium reduction in vitro and in vivo. Physiological analysis of a mutant of D. desulfuricans with a mutation of the gene encoding the type 1 tetraheme cytochrome c3 had demonstrated that uranium reduction was negatively impacted while sulfate reduction was not if lactate were the electron donor. This was thought to be due to the presence of a branched pathway of electron flow from lactate leading to sulfate reduction. Our experimental plan was to elucidate the structural and mechanistic details of uranium reduction involving cytochrome c3

Genes for Uranium Bioremediation in the Anaerobic Sulfate-Reducing Bacteria

Objective A: Electron transfer components necessary for uranium reduction. Objective B: Possible FNR-analog in the sulfate-reducing bacteria. Attempts to isolate FNR or FIKJ analogs from Desuflovibrio through the design of degenerate primers for amplification of portions of the genes has not been successful. In contrast, several amplicons have been generated for the genes encoding the regulators of two-component signal sequences. Since several global regulators fall into this class, we are attempting to obtain sufficient sequence information to indicate what metabolic pathways are affected by the regulators. Cloning and sequencing of two such amplicons has revealed that bona fide two-component regulators are present in Desulfovibrio

Genes for Uranium Bioremediation in the Anaerobic Sulfate-Reducing Bacteria

The objectives of the previous grant period were designed to explore the electron transport pathway employed by the sulfate-reducing bacteria (SRB) for the reduction of U(VI) to U(IV). More specifically experiments were designed to determine whether U(VI) reduction by members of the genus Desulfovibrio was mediated by a unique, dedicated reductase or occurred as a fortuitous reaction with a reductase naturally involved in alternative reduction processes. In addition, the regulation of the hierarchical expression of terminal electron acceptors (reductases) in the SRB was to be examined