Source: UNIVERSITY OF MISSOURI submitted to
GENETICS OF SULFATE-REDUCING BACTERIA APPLIED TO MECHANISMS OF BIOCORROSION AND BIOREMEDIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0155987
Grant No.
(N/A)
Project No.
MO-BCSL0071
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2000
Project End Date
Jun 30, 2006
Grant Year
(N/A)
Project Director
Wall, J. D.
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
BIOCHEMISTRY
Non Technical Summary
The ubiquitous sulfate-reducing bacteria affect each citizen because of the corrosive nature of their metabolism on iron metals in soils and waters. Each of us has smelled the diagnostic indicator of their activity, the "rotten egg" odor, in contaminated environments. Now evidence is accumulating that this metabolic activity might be put to a beneficial use for bioremediation. We propose to learn how these bacteria make energy so that we might both control and use these microbes. Collaborators at the University of Oklahoma are also involved in these efforts.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1334010100015%
1334010101010%
1334010104010%
1334010108015%
1334099100015%
1334099101010%
1334099104010%
1334099108015%
Goals / Objectives
The sulfate-reducing bacteria of the genus Desulfovibrio play critical roles in environmental metal corrosion and may provide important mechanisms for toxic metal remediation. We propose to explore the energy generating pathways of these bacteria to facilitate their control and utilization. To analyze these pathways, we will use genetic approaches that will require the development of a system for marker-exchange mutagenesis, an efficient tool for random transposon mutagenesis, and a controlled-expression vector for Desulfovibrio. These tools will then be applied to elucidate the roles of the multiple hydrogenases and cytochromes that are present. First we will isolate the genes for the various hydrogenases and c-type cytochromes, attempt to create mutations (optimally deletions), and examine the physiological affects of the mutations. In addition, the regulation of each of these genes or their operons will be explored through promoter and protein fusions. Finally, we propose to explore the effects of each of the mutated electron transport components on uranium metabolism, with the goal of identifying the electron pathway to uranium, the uranium reductase, and its regulation.
Project Methods
The genetic tools will be developed to pursue the physiological studies. First, a thyA deletion mutant of D. desulfuricans G20 will be created to allow selection both for and against vectors containing this gene in the deletion strain. Secondly, to increase the efficiency of transposon mutagenesis, the facility of the newly described "transposasome" will be explored. Third, to make mutations in essential genes and explore the physiological role of the gene products, a regulated promoter that can be used to control expression of genes tightly will be needed. Available systems useful in other bacteria will be tested. The genome sequencing project of Desulfovibrio allows a first approach to the isolation of additional hydrogenase and c-type cytochrome genes in our strain that will complement the work already in hand. The thyA marker-exchange system will allow mutations to be created in these genes that do not result in the inclusion of antibiotic markers in the chromosome, facilitating the construction of multiple mutations. Single-copy promoter fusions will be created for each gene for the study of regulation with different substrates and stages of growth. Suppressors of mutations that block electron flow should identify interacting protein partners or alternative pathways. To obtain evidence for an involvement of electron transfer components in uranium reduction, the effects of mutations in various components on U(VI) reduction will be examined. Physiological changes in bacteria exposed to U(VI) will also be explored to understand the limitations to the application of bacteria to U(VI) sequestration as U(IV). Mutants generated by random transposon insertion will be assayed for an alteration in U(VI) reduction.

Progress 07/01/00 to 06/30/06

Outputs
OUTPUTS: To unravel the energy generation processes of the sulfate-reducing bacteria of the genus Desulfovibrio, we have joined a collaborative effort with the Virtual Institute for Microbial Stress and Survival (primarily located at the Lawrence Berkeley National Laboratory). Our role in the collaboration is to make deletions of the genes of Desulfovibrio vulgaris Hildenborough (DvH), tagged the proteins through gene modifications, and interpret the physiological findings. We have generated >30 deletions of genes or operons in DvH. We have also identified the location of 100 transposon-generated mutations. For protein tagging, we have created seven confirmed gene modifications that express tandem tagged proteins. In addition we have another 60 tagging constructs in the pipeline for testing. We are creating a exometabolome for DvH fermenting pyruvate. PARTICIPANTS: Barbara Giles (Lab manager, performed all work with Desulfovibrio desulfuricans G20 genetics). Huei-Che Yen (Post doctoral fellow, physiology of DvH). Grant Zane (Research Specialist, creates deletions of DvH). Jarrod Robertson (Research Specialist, creates deletions of DvH). Kelly Bender (Post doctoral student, created deletions of global regulators). Leena Pattarkine (Post doctoral student, purified and crystallized cytochrome c3). Kimberly Keller (Post doctoral student, in training, created the inframe deletion system for DvH). Dwayne Elias (Research Assistant Professor, oversees gene tagging project). Tom Juba (Research Specialist, transforms the tagging contructs into DvH). Matthew Shirley (Undergrad, sequences the sites for transposon interruption). Elliott Drury (MS student, studied the potential roles of proteins annotated to be hypothetical in the genome). At Lawrence Berkeley National Laboratory Terry Hazen Swapnil Chhabra Mark Biggin At University of California, Berkeley Adam P. Arkin Jay Keasling At University of Washington, Seattle David Stahl Chris Walker Segey Stolyar Professional training: Kimberly Keller, Kelly Bender, Leena Pattarkine, Dwayne Elias, Elliott Drury, Ray Payne, Christopher Hemme, Matthew Shirley, Kate Hart, and Suzanne Miller TARGET AUDIENCES: Audiences: Undergraduate students in Biochemistry, Researchers in related areas in environmental microbiology, and DOE PIs and program directors. Efforts: Teaching Invited seminars Presentations at American Society for Microbiology Presentations at EU-US Environmental Biotechnology events Presentations at Genomics:GTL PI Workshops PROJECT MODIFICATIONS: This is a termination because the last application was denied.

Impacts
The following two examples of deletions created in our lab serve to illustrate our role in the physiological interpretations. We have created deletions by marker exchange mutagenesis to explore the electron transport pathways of DvH. Energy generation by sulfate respiration coupled to the oxidation of hydrogen is dependent on the delivery of electrons from the periplasmic hydrogenases to the enzymes for reduction of sulfate. We have created deletions of two transmembrane complexes encoded by DVU2404-DVU2399 and DVU0848-DVU0851 that are annotated as Heterodisulfide reductase/hydrogenase (Hdr) and Quinone-interacting membrane-bound oxidoreductase (Qmo), respectively. The mutants have now been explored for their ability to support sulfate reduction by hydrogen and by the organic acid, lactate. There is no difference between the wild type strain and the Hdr mutant for growth with these substrates. We infer that this complex is either not on the pathway explored or that the electrons have readily accessible alternative pathways. For the Qmo deletion, the mutant is completely unable to use sulfate as the terminal electron acceptor but can reduce sulfite just like wild type. We have seen no compensation or suppression of the growth defect on sulfate which we interpret to mean that the Qmo complex is solely responsible for delivering electrons to the Adenylylsulfate reductase for the first, two electron reduction of sulfate. We have made the plasmid vectors for use in generating inframe deletions in DvH so that genes within operons may be targeted without concern for polarity. This procedure will also allow tagging of proteins by gene modification of genes within operons. We are targeting a Type 1 restriction endonuclease to test the system and to provide a host bacterium with reduced DNA restriction for transformations. In collaboration with Morgan Price at the University of California, Berkeley, we have also been exploring the pathway for methionine biosynthesis. The genome sequence of DvH did not reveal orthologs for the biosynthetic enzymes. Because the bacterium is clearly not a methionine auxotroph, alternative enzymes must perform the critical steps. Morgan has made bioinformatics predictions of candidate genes for the missing steps and we have deleted two of the predicted genes. Unfortunately, the deletion strains still do not require methionine. Thus we have not yet identified the critical genes or enzymes for this pathway.

Publications

  • Bender, K. S., H.-C. Yen, and J. D. Wall. 2006. Analyzing the metabolic capabilities of Desulfovibrio species through genetic manipulation, p. 157-174. In S. E. Harding (ed.), Biotechnology and genetic engineering reviews. Vol 23. Lavoisier, France.
  • Pattarkine, M.V., J.J. Tanner, C.A. Bottoms, Y.-H. Lee, and J.D. Wall. 2006. Desulfovibrio desulfuricans G20 tetraheme cytochrome structure at 1.5 A and cytochrome interaction with metal complexes. J. Mol. Biol. 358(5):1314-1327.
  • Wall, J.D., and L.R. Krumholz 2006. Uranium reduction. Annu Rev Microbiol. 60:167-185.
  • Walker, C.B., S.S. Stolyar, N. Pinel, H.C.B. Yen, Z. He, J. Zhou, J.D. Wall, and D.A. Stahl. 2006. Recovery of temperate Desulfovibrio vulgaris bacteriophage using a novel host strain. Environ. Microbiol. 8:1950-1959.
  • Mukhopadhyay, A., Redding, A. M., Joachimiak, M. P., Arkin, A. P., Borglin, S. E., Dehal, P. S., Chakraborty, R., Geller, J. T., Hazen, T. C., He, Q., Joyner, D. C., Martin, V. J. J., Wall, J. D., Yang, Z. K., Zhou, J., Keasling, J. D. (2007). Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 189: 5996-6010
  • Stolyar, S., He, Q., Joachimiak, M. P., He, Z., Yang, Z. K., Borglin, S. E., Joyner, D. C., Huang, K., Alm, E., Hazen, T. C., Zhou, J., Wall, J. D., Arkin, A. P., Stahl, D. A. (2007). Response of Desulfovibrio vulgaris to alkaline stress. J. Bacteriol. 189: 8944-8952
  • Bender, K.S., H.C.B. Yen, C.L. Hemme, Z. Yang, Z. He, J. Zhou, K.H.Huang, E.J. Alm, T.C. Hazen, A.P. Arkin, and J.D. Wall. 2007. Analysis of a ferric uptake regulator (Fur) mutant of Desulfovibrio vulgaris Hildenborough. Appl. Environ. Microbiol. 73:5389-5400.


Progress 01/01/05 to 12/31/05

Outputs
The ubiquitous sulfate-reducing bacteria affect each citizen because of the corrosive nature of their metabolism on iron metals in soils and waters. Each of us has smelled the diagnostic indicator of their activity, the "rotten egg" odor, in contaminated environments. Now evidence is accumulating that this metabolic activity might be put to a beneficial use for bioremediation. We propose to learn how these bacteria make energy so that we might both control and use these microbes. We have developed a procedure for the tagging of proteins in the sulfate-reducing bacteria. These tags will serve as "handles" for the gentle isolation of the target proteins and, it is hoped, the proteins that form functional complexes with the target. The identification of the co-isolated proteins will provide the beginning of an understanding of molecular machines in the sulfate reducers and how these machines may be changed during stress. We have also developed a procedure for the generation of deletion mutations of the sulfate-reducing bacteria of the genus Desulfovibrio. Using this procedure we have created deletions of the global regulators Fur, PerR, and Zur as well as a number of ion transport genes. The characterization of these mutants forms the basis of continued work to elucidate the regulatory systems of these bacteria.

Impacts
The ability to predict the fate of toxic metals and radionuclides in contaminated environments is a necessary objective for remediation. Our work will contribute to a more accurate picture of the ability of microbes to interact with these contaminants and to be influenced by augmentation during bioremediation efforts. All microbes are limited by one or a number of nutitional or physical factors in the environment. We are elucidating the response of the metal-metabolizing sulfate-reducing bacteria to environmental stresses to be able to predict their versatility and robustness in contaminated environments.

Publications

  • Miller, W.H., P. Duval, S.S. Jurisson, J.D. Robertson, J.D. Wall, T.P. Quinn, W.A. Volkert, and G.M. Neumeyer. 2005. Radiochemistry at the University of Missouri-Columbia: A joint venture with chemistry, nuclear engineering, molecular biology, biochemistry, and the Missouri. University Research Reactor (MURR). J. Radioanal. Nuc. Chem. 263:131-136.
  • Mukhopadhyay, A., He, Z., Yen, H.-C., Alm, E.J., He, Q., Huang, K.H., Baidoo, E.E., Chen, W., Borglin, S.C., Redding, A., Holman, H.-Y., Sun, J., Joyner, D.C., Keller, M., Zhou, J., Arkin, A.P., Hazen, T.C., Wall, J.D., Keasling, J.D. Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J. Bacteriol. 2006 188: 4068-4078.
  • 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 2006. Global Analysis of Heat Shock Response in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 2006 188: 1817-1828.


Progress 01/01/04 to 12/31/04

Outputs
We have obtained spectroscopic information for a functional interaction of type I tetraheme cytochrome c3 purified from the sulfate-reducing bacterium, Desulfovibrio desulfuricans G20, and uranium. In vitro, this cytochrome c3 is a uranium reductase. We have obtained crystal structure of the oxidized cytochrome refined to the 1.5 Angstrom level, obtained the structure of a crystal reduced with sodium dithionite, and attempted to determine the site of interaction of Mo(VI) with the protein in the crystals. Attempts to find a site of binding of U(VI) to the protein have been unsuccessful to date.__ A Desulfovibrio desulfuricans G20 mutant deleted in the gene for the dominant cytochrome c3 has been obtained. Its phenotype is essentially like that of the mutant generated by plasmid interruption of the cycA gene. Currently we are attempting to complement the mutations to confirm that mutation of the cycA gene is solely responsible for the alterations in metal reduction and cell growth observed.__ Site directed mutations of the cycA gene have been constructed. They include F19A, Y27A, C45A, K66A, K72A, and M80K. The Y27A mutation rendered the protein sufficiently unstable such that we were unable to obtain mutant protein from the Escherichia coli recombinant strain. While we do not have kinetic data for this process as yet, all the mutant proteins are still able to reduce U(VI) to U(IV).__ As a part of an examination of environmental stresses on the metabolism of the sulfate reducing bacteria, we have explored the effects of salt stress on Desulfovibrio vulgaris Hildenborough. Curiously sodium and potassium effects are additive unlike enteric bacteria where potassium can alleviate the negative consequences of high sodium concentrations. Cells elongate in the presence of 450 mM sodium ion and slow their growth rate by about 50%. When such cells are supplemented with 2 mM glycine betaine the elongation and growth impairment are almost completely reversed. This work is being complemented with a transcript analysis of sodium stressed cells.__ Deletion mutation procedures have been established for these sulfate-reducing bacteria. A four step PCR approach is used to generate a mutagenic cassette containing DNA regions from upstream and downstream of the gene to be deleted now flanking an antibiotic resistance gene. After capture of the cassette in a cloning vector, the recombinant plasmid is introduced into the Desulfovibrio strains by electrotransformation and antibiotic resistance is selected. Double recombinants that have resulted in marker exchange are then isolated and identified by diagnostic PCR. Using this procedure we have begun to generate mutations of global regulatory genes, sodium transporters, and cation diffusion facilitators. Characterization of these mutant strains is underway.__ Mutants with specific phenotypes are being sought from the transposon library. Location of the transposon in a large number of mutants is a near term goal.

Impacts
The ability to predict the fate of toxic metals and radionuclides in contaminated environments is a necessary objective for remediation. Our work will contribute to a more accurate picture of the ability of microbes to interact with these contaminants and be influenced by augmentation during bioremediation efforts.

Publications

  • Hemme, C.L., and J.D. Wall. 2004 Genomic insights into the gene regulation of Desulfovibrio vulgaris Hildenborough. Omics 8:1-13.
  • Wall, J.D. 2004. Rain or shine-a phototroph that delivers. Nature Biotechnol. 22:40-41.
  • Payne, R.B., L.Casalot, T.Rivere, J.H. Terry, and J.D. Wall. 2004. Interaction between uranium and the cytochrome c3 of Desulfovibrio desulfuricans strain G20. Arch. Microbiol. 181:398-406.
  • Heidelberg, J.F., R. Seshadri1, S.A. Haveman, C.L. Hemme, et al. 2004. The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough: consequences for its energy metabolism and reductive metal bioremediation. Nature Biotech. 22:554 - 559.
  • Payne, R.B., C.L. Hemme, and J.D. Wall. 2004. A new frontier in genomic research. World Pipelines 4:53-5


Progress 01/01/03 to 12/31/03

Outputs
The ability of the sulfate-reducing bacterium (SRB) Desulfovibrio desulfuricans G20 to reduce the toxic radionuclide uranium from an oxidation state of six to four brings about a change in the solubility of the resulting complex. U(VI) is found in aerobic environments as UO2 with two positive charges and the reduced form, U(IV) is UO2, not charged, and therefore quite insoluble. The crystals formed from the latter precipitate as the mineral uraninite and can be filtered from aqueous solutions. This offers a possible bioremediation procedure for contaminated groundwater. Thus electron flow in the SRB is critical to the reduction of U(VI) and has been the object of our investigations. 1) We have found that sodium gradients (that are an energy currency in bacteria) may result from electron flow in G20. If so, the sodium content of the aqueous environments may make a significant difference in the predicted bioremediation potential. How this occurs and what the contribution is to the energy budget forms a future goal. 2) The complete genome sequence of both Desulfovibrio vulgaris Hildenborough and G20 are now available and regulatory motifs continue to be sought through computational approaches. 3) The number and location of all multiheme c-type cytochromes have been iterated. The functional roles of these proteins are being sought by seeking information about expression through Northern analyses. 4) Purified cytochrome c3 has been shown to be oxidized by U(VI) in the test tube. Thus this protein is a good candidate for the reductase. Site-directed mutations of the gene for this protein have been constructed and will be tested for retention of the ability to function in U(VI) reduction. 5) The array of electron donors and terminal electron acceptors of D. vulgaris and D. desulfuricans has been explored. It appears that both strains readily ferment and likely grow more robustly by fermentation or by respiring fumarate than by sulfate respiration. 6) Efficient transposon mutagenesis has been documented and a collection of over fifty different mutations have been examined to determine the exact insertion site of the transposon.

Impacts
The ability to predict the fate of toxic metals and radionuclides in contaminated environments is a necessary objective for remediation. Our work will contribute to a more accurate picture of the ability of microbes to interact with these contaminants and be influenced by augmentation during bioremediation efforts.

Publications

  • Wall, Judy D., Christopher L. Hemme, Barbara Rapp-Giles, Joseph A. Ringbauer, Jr., Laurence Casalot, and Tara Giblin. 2003.Genes and Genetic Manipulations of Desulfovibrio. In L.G. Ljungdahl et al. (eds.) Biochemistry and physiology of anaerobic bacteria. Springer, New York, pp. 85-98.
  • Hemme, C.L., and J.D. Wall. 2004 Genomic insights into the gene regulation of Desulfovibrio vulgaris Hildenborough. Omics (in press).
  • Payne, R.B., L.Casalot, T.Rivere, J.H. Terry, and J.D. Wall. 2004Interaction between uranium and the cytochrome c3 of Desulfovibrio desulfuricans strain G20 (Arch. Microbiol., in press).
  • Rivere, Tessa. Reduction of Uranium by Sulfate-reducing Bacteria, .In Partial Fulfillment of the Requirements for the Master of Science Degree in Nuclear Engineering, Dec. 2002. Drs. Miller and Wall, Advisors.


Progress 01/01/02 to 12/31/02

Outputs
Earlier work in the lab showed that the predominant tetraheme c-type cytochrome was a component of the electron flow to uranium in D. desulfuricans G20 (Payne et al., 2002). Cells were grown in the presence of uranyl acetate to explore whether this cytochrome was induced in expression (Payne et al., in preparation). Counter to expectations, the ability of the cells for U(VI) reduction with organic acids as electron donors was actually decreased by previous exposure to U(VI). To account for this decrease, an attempt was made to determine whether the cytochrome c3 content was normal. Unexpectedly cytochrome c3 was not found in periplasmic or whole cell extracts of D. desulfuricans G20 grown in the presence of 1 mM uranyl acetate. It was found that the predominant tetraheme cytochrome was binding tightly and quantitatively to the precipitated uraninite that was being removed from the extracts with cell debris. The association of the cytochrome with the uraninite was not disrupted by 1 M NaCl, although clearly the loading buffer for immunoblots did remove the protein. The adsorption of the cytochrome to the insoluble metal complex has been interpreted to be non-specific, since the protein also adsorbed to commercially available UO2, CuO, Fe2O3, and chemically prepared CuS. This result would argue against the possibility that cytochromes of Desulfovibrio might be used as external electron shuttles to insoluble substrates in the environment. To further explore the relationship between the cellular exposure to U(VI) and the expression of cytochrome c3, a translational fusion was constructed between the start codon of cycA and beta-galactosidase. Homologous recombination with the cycA-upstream DNA creating strain G25 containing a beta-galactosidase fusion under the control of the wild-type cycA promoter in a cellular background that was wild-type for cytochrome c3. Growth of D. desulfuricans in the presence of uranyl acetate had no detectable effect on the expression of cycA. The availability of the complete genome sequence of Desulfovibrio vulgaris Hildenborough (The Institute for Genome Research, John Heidelberg, contact person) altered our approach to clues about the regulatory circuitry of the SRB. A computational strategy was adopted from George Church and coworkers for detecting conserved regulatory motifs in the upstream regions of ORFs in bacterial genomes using the AlignACE suite of programs. This program returned a set of putative motifs that were then subjected to statistical analysis. Not only should conserved motifs be revealed but also motifs unique to Desulfovibrio should be identified. Of course, laboratory confirmation of all motifs is still necessary. To explore a possible involvement of nitrite reductase in the reduction of U(VI) in D. desulfuricans, experiments were carried out to determine whether nitrite would compete for electrons available for U(VI) reduction. Sodium nitrite at 1 mM did not detectably inhibit U(VI) reduction by wild-type G20 when the source of reductant was 10 mM lactate, 10 mM pyruvate, or one atmosphere of hydrogen gas.

Impacts
The robustness of the ability of common soil bacteria (sulfate-reducing bacteria) to change the solubility of uranium in ground waters is being elucidated. The development of a logical approach to bioremediation of heavy metal contaminated environments is still a future goal.

Publications

  • Payne, Rayford B., and Judy D. Wall. 2002. Effect of Uranium on the cytochrome c3 of Desulfovibrio desulfuricans. Abstr. Amer. Soc. Microbiol. 102nd Gen. Meet., Salt Lake City, Utah, p. 448.
  • Hemme, Christopher L., and Judy D. Wall. 2002. Predicting the Genetic Regulation of Uranium Reduction in Desulfovibrio vulgaris. 2nd Annual TIGR-ASM Conference on Microbial Genomes, Los Vegas, NV.
  • Payne, Rayford B., Darren M. Gentry, Barbara J. Rapp-Giles, Laurence Casalot, and Judy D. Wall. 2002 Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant. Appl. Environ. Microbiol. 68:3129-3132.


Progress 01/01/01 to 12/31/01

Outputs
The sulfate-reducing bacterium, Desulfovibrio desulfuricans strain G20, is being examined for the electron transfer pathways for energy generation. These same pathways are expected to be critical for the bacterium to metabolize metals, resulting in biocorrosion of ferrous metals or bioremediation of toxic metals. We have determined that the tetraheme cytochrome c3 is the primary route for electrons from hydrogen to toxic heavy metals such as uranium. An analysis of a mutant in the gene encoding the primary tetraheme cytochrome shows that the electrons from lactate and those from pyruvate follow different pathways to sulfate. Those from pyruvate appear to flow through hydrogen in support of the hydrogen cycling model of Odom and Peck for Desulfovibrio. Studies of the regulation of the tetraheme cytochrome c3 reveal an essentially constitutive expression.

Impacts
An anlysis of the energy generating mechanisms of the sulfate-reducing bacteria may permit intervention and limitation of the corrosion of metals by these microbes in the environment.

Publications

  • Casalot, L., B. J. Rapp-Giles, and J. D. Wall. 2001. Regulation of cycA in Desulfovibrio desulfuricans G20. Abstr. 101 Gen. Meet. Amer. Soc. Microbiol., Orlando, FL, p. 610.


Progress 01/02/00 to 12/31/00

Outputs
The sulfate-reducing bacterium, Desulfovibrio desulfuricans strain G20, is being examined for the electron transfer pathways for energy generation. These same pathways are expected to be critical for the bacterium to metabolize metals, resulting in biocorrosion of ferrous metals or bioremediation of toxic metals. To that end, a mutation has been created in the gene encoding the most abundant c-type cytochrome, c3. This mutant demonstrates that this is not an essential cytochrome. The ability of the mutant bacterium to reduce the soluble U(VI) to the much less soluble U(IV)is decreased by at least 50% suggesting that cytochrome c3 is in the electron pathway to U(VI) or the actual reductase. Additional electron pathways or reductases are obviously present. Preliminary genome sequences of a related sulfate reducer reveal the presence of multiple tetraheme cytochrome genes in which we are now trying to create mutations. These will allow us to explore the physiological function(s) of these cytochromes and to determine their role, if any, in the reduction of toxic metals.

Impacts
Understanding the biochemical mechanism of toxic metal metabolism by the sulfate-reducing bacteria will increase the possibility of predicting the outcome of biostimulation of remediation efforts in a given contaminated environment.

Publications

  • Rapp-Giles, B. J., L. Casalot, R. S. English, J.A. Ringbauer, Jr., A. Dolla, and J.D. Wall. 2000. Cytochrome c3 mutants of Desulfovibrio desulfuricans. Appl. Environ. Microbiol. 66:671-677.
  • Ringbauer,Jr., J. A. Hydrogenases of Desulfovibrio desulfuricans G20, Ph.D. Dissertation, December 2000, Biochemistry Department, University of Missouri-Columbia


Progress 01/01/99 to 12/31/99

Outputs
We have been successful in constructing a mutation in the gene encoding cytochrome c3 in the sulfate-reducing bacterium, Desulfovibrio desulfuricans. Although this cytochrome accounts for >80 % of all c-type cytochrome in these bacteria, it appears not to be essential for all growth conditions. A major defect in the mutant is an inability to grow well with pyruvate as the source of reductant. Apparently, this cytochrome interacts with pyruvate dehydrogenase or the ferredoxin shown to act as electron acceptor of PDH in vitro. When the mutant is incubated with pyruvate, hydrogen is produced suggesting that the reductant from pyruvate is redirected in the absence of the cytochrome. With polyclonal antibodies to cytochrome c3, Western analysis has revealed that this cytochrome is increased in expression as cells grown on lactate-sulfate medium enter stationary phase. This unexpected result suggests that this cytochrome is not constitutively expressed but subject to regulation, possibly at transcription. The region of DNA upstream of the gene has an apparent SRB promoter; however, RT-PCR evidence suggests there are transcripts that begin farther upstream than that promoter. The promoter, or promoters, will be identified by S1 mapping and primer extension and confirmed by mutagenesis. Regulation will be examined by protein fusions with beta-galactosidase. Plasmid constructs will be used that introduce the reporter gene into the chromosome in single copy under the regulation of the wild-type promoter. We have already prepared one such construct with pVK111 and are attempting to integrate the construct. Uranium reduction by D. desulfuricans G20 has been demonstrated. The cytochrome c3 mutant reduces uranium still but at about 50% of the rate of the wild type. Curiously wild-type cells that are grown in the presence of 1 mM uranium have no detectable cytochrome c3. We are exploring the possible causes of that loss.

Impacts
Our findings impact the potential practical application of the sulfate-reducing bacteria to bioremediation of uranium.

Publications

  • Rapp-Giles, B.J.,Casalot, L., English, R.S., Ringbauer, Jr., J.A., Dolla, A., and Wall, J.D. 2000. Cytochrome c3 mutants of Desulfovibrio desulfuricans. Appl. Environ. Microbiol. 66:671-677.


Progress 01/01/98 to 12/31/98

Outputs
During 1998, 1) An [Fe] hydrogenase operon was cloned from Desulfovibrio desulfuricans G20. 2) A mutant with a disruption in this operon was constructed. 3) No significant phenotype has been associated with the mutation. 4) Activity staining of separated protein extracts confirmed the absence of the major cellular hydrogenase activity. 5) Sequencing of revertants of an insertional mutation in the gene for cytochrome c3 revealed DNA rearrangments. From these studies, we conclude that this protein plays an important metabolic role apart from accepting electrons from the [Fe] hydrogenase. 6) G20 is capable of reducing U(VI) to U(IV). 7) D. desulfuricans Essex 6 has been shown to be genetically accessible, since we have successfully electrotransformed this strain and begun to create transposon mutants.

Impacts
(N/A)

Publications

  • M. Rousset, L. Casalot, P. de Phillip, Z. Dermoun, B.J. Rapp-Giles, J.P. Belaich, and J.D. Wall. 1998. Construction of new cloning vectors for sulfate-reducing bacteria and their use. Plasmid 39:114-122.
  • Rapp-Giles, B.J., J.D. Wall. 1998. Genetics of cytochrome c3 from Desulfovibrio desulfuricans G20. 98th Gen. Meet. Amer. Soc. Microbiol. Atlanta, Georgia, May 17-21, K-61
  • Ringbauer, Jr., J.A., and J.D. Wall. 1998. Hydrogenases of Desulfovibrio desulfuricans G20: Roles and regulation. . 98th Gen. Meet. Amer. Soc. Microbiol. Atlanta, Georgia, May 17-21, K-60.


Progress 01/01/97 to 12/31/97

Outputs
Progress continues with the examination of hydrogen metabolism in the sulfate-reducing bacterium (SRB), Desulfovibrio desulfuricans. Genes for (NiFe) and (Fe) hydrogenases have been cloned. A mutant with genes for the (NiFe) hydrogenase has been constructed and a similar mutation in the (Fe) hydrogenase genes is in progress. The cytochrome c3 encoding gene cycA has been cloned and attempts to mutagenize it by plasmid insertion have apparently failed. Regulation studies show that iron limitation represses expression of the cytochrome gene. A fragment containing 163 bp upstream of the start codon for cycA exhibited sigma-70 like promoter sequences and supported transcription in Escherichia coli but did not do so in D. desulfuricans. Longer stretches of upstream sequence are now being examined for promoter activity in the SRB. Additional cloning vectors and promoter probe vectors have been constructed to carry out these experiments.

Impacts
(N/A)

Publications

  • AUBERT, C., et al. 1997. A single mutation in the heme 4 environment of Desulfovibrio desulfuricans Norway cytochrome c3 (Mr26,000) greatly affects the olecule reactivity. J. Biol. Chem.
  • ROUSSET, M., L. CASALOT, B.J. RAPP-GILES, Z. DERMOUN, P. DE PHILIP, J.-P. 1997. In press. Bs for cloning in Desulfovibrio. 1997. Plasmid.
  • WICKMAN, T. 1997. A physical and genetic map of the chromosome of the ulfate-reducing bacterium Desulfovibrio desulfuricans G20. Ph.D. Dissertation. University of Missouri- Columbia.


Progress 01/01/96 to 12/30/96

Outputs
During 1996, 1) the physical map of Desulfovibrio desulfuricans has been refined, genetic markers added and cosmid coverage of some regions accomplished. 2) The 16S rDNA has been sequenced and the phylogeny of D. desulfuricans strain G100A explored. 3) Genes encoding a NiFe hydrogenase have been isolated and partially sequenced. 4) A mutation of the hydrogenase large subunit gene has been constructed and phenotypes of the mutant are being sought. 5) The cytochrome c3 gene has been cloned and sequenced. 6)A putative promoter region of the c3 gene has been cloned into a promoter probe vector for transcriptional analysis. 7) Additional vectors have been created that allow blue/white selection in E. coli and mobilization and stable inheritance in the sulfate-reducing bacteria (SRB). 8) Conditions to visualize color development from the beta-galactosidase substrate X-gal with colonies of the SRB have been obtained.

Impacts
(N/A)

Publications

  • WALL, J.D., T. MURNAN, J. ARGYLE, R.S. ENGLISH, and B.J. RAPP-GILES. 1996. Transposon mutagenesis in Desulfovibrio desulfuricans: Development of a random mutagenesis tool from Tn7. Appl. Environ. Microbiol. 62:3762-3767.
  • WICKMAN, T., and J.D. WALL. 1996. Correlation of the physical map of Desulfovibrio desulfuricans G201 with a preliminary genetic map. Abstr. 96th Gen. Meet. Amer. Soc. Microbiol., New Orleans, p. 491.
  • RAPP-GILES, B. J., and J.D. WALL. 1996. Cytochrome c3 from Desulfovibrio desulfuricans strain G200. Abstr. 96th Gen. Meet. Amer. Soc. Microbiol., New Orleans, p. 558.
  • ENGLISH, R. S., and J.D. WALL. 1996. Construction and use of a promoter-probe vector in the sulfate-reducing bacteria. Abstr. 96th Gen. Meet. Amer. Soc. Microbiol., New Orleans, p. 511.


Progress 01/01/95 to 12/30/95

Outputs
Genetics of the sulfate-reducing bacteria continues to be the focus of the laboratory for the purpose of examining the metabolic roles of this strictly anaerobic bacterium in the environment. A random mutagenesis tool has been developed from a mutant Tn7 transposon. Tests are currently underway to increase the efficiency of transposition in these bacteria by augmenting the concentration of one polypeptide of the transposase. Over twenty transpositions have been mapped by CHEF gels, confirming the random' nature of the insertions and the physical map of the chromosome. A promoter probe vector was created, a library generated, and comparisons of the expression of the reporter gene in E. coli versus Desulfovibrio carried out. A strongly expressed promoter in Desulfovibrio has been sequenced and shown to be a ribosomal protein gene. A cluster of ribosomal proteins has been observed. Genes for cytochrome c3 and a (NiFe) hydrogenase have been isolated and sequenced.

Impacts
(N/A)

Publications

  • BORGHESE, R., AND J.D. WALL. 1995. Regulation of expression of the glnBA operon of Rhodobacter capsulatus. J. Bacteriol. 177:4549-4552.


Progress 01/01/94 to 12/30/94

Outputs
Transposon mutagenesis of the sulfate-reducing bacteria (SRB) continues to be a goal of the laboratory and none of our observations regarding transposition have yet been published. The successful transposition of Tn7 into a unique site in the chromosome was confirmed. The two plasmid mini Tn7-based system for the single-copy insertion of cloned genes designed by Gary Roberts and coworkers was shown to be useful in the SRB. The insertion site has been shown by sequencing to be very similar to that used in other Gram-negative bacteria and to be a neutral site. To create a transposon that may integrate randomly and be useful for mutagenesis, we have interrupted the tnsD gene responsible for recognition of the unique site. Tests are in progress to determine the insertion sites of the mutant Tn7. We are also attempting to make a library of random insertion mutants via homologous recombination rather than by transposition. The sacB cassette is being employed to capture transposons in Desulfovibrio desulfuricans G20 for the possible creation of genetic tools and to eliminate ambiguities caused by repeated DNA in the physical map. A tentative linkage map of SpeI and NheI fragments of the D. desulfuricans G20 chromosome has been constructed. Double digestions of fragments from the CHEF gels are being used to confirm the predictions of the alignments. Finally, a number of shuttle vectors have now been constructed from the endogenous plasmid pBG1, and several are being used routinely in the lab.

Impacts
(N/A)

Publications


    Progress 01/01/93 to 12/30/93

    Outputs
    See MO-BCHC0072.

    Impacts
    (N/A)

    Publications

    • No publications reported this period.