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Mechanisms of U(VI) Reduction and Sediment Growth in Desulfovibrio

This study aims to identify the mechanisms of survival and U(VI) reduction in sediment bacteria, specifically Desulfovibrio G20. Through mutagenesis and growth experiments, potential genes and functions involved in U(VI) reduction and response to mutagens will be identified. The study also explores the role of specific genes in drug efflux and DNA repair. Acknowledgments to DOE ERSP program and researchers involved.

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Mechanisms of U(VI) Reduction and Sediment Growth in Desulfovibrio

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  1. Mechanisms of U(VI) Reduction and Sediment Growth in Desulfovibrio Lee Krumholz Department of Botany and Microbiology

  2. Goals • Identify mechanisms of survival of sediment bacteria in U(VI) contaminated sediments. • This was further refined to • Identification of mechanisms of dealing with U(VI) • Identification of mechanisms of survival in Anoxic sediments.

  3. Desulfovibrio G20 • Isolated from an oil producing reservoir. • 4.1 Mb, 3598 candidate genes • Doubling time is ~4.6 hours

  4. Mechanisms of U(VI) reduction

  5. Identification of Genes involved in U(VI) Response

  6. 10 mutants

  7. Potential Functions of mutated genes in U(VI) Sensitive Mutants • DNA repair • rRNA methylation • Protein Renaturation

  8. Growth Experiment with B11E9 Washed Cell U(VI) reduction test by 24 mutants

  9. The Region surrounding the mutation in B11E9 D. vulgaris

  10. Mechanism of As(VI) Reduction

  11. Loss of As(V) Tolerance. • B11E9 also lost As(V) resistance when grown in lactate sulfate media with 20mM As(V)

  12. Growth and U(VI) reduction of G20 with Cd

  13. Mechanism of U(VI) Reduction

  14. U(VI) Reduction by E. coli transformant

  15. Signature Tagged Mutagenesis • First developed to identify virulence genes in pathogens. • Use the technique to identify functions involved in sediment growth. Different from functions needed for lab growth.

  16. Unique tag carried on transposon Tagged transposon moved into bacteria Recover viable cells and amplify tag region Mutants assembled in microtiter dishes Sediment incubations Pooled liquid culture PCR generation of tagged probes and hybridization to complimentary tag target Replicate tags onto solid surface (membrane) Overview of signature tagged mutagenesis (STM)

  17. A6(pA2) 1.0E+07 G9(pB1) B11(pC2) 1.0E+06 A3(pE2) C12(pG2) cell number 1.0E+05 H6(pG2) B11(pF2) 1.0E+04 pool C2 B8(pE1) 1.0E+03 0 5 10 15 20 Growth of potential non-survival mutants of Desulfovibrio in sediment Time (day)

  18. Screened ~5000 mutants for each bacterium by STM and identified 100 mutants in G20 and 46 mutants in MR-1 Recurring Themes DNA repair Transcriptional regulators Phage-related proteins Transport proteins (multidrug efflux) Conserved and hypothetical proteins

  19. DNA repair •sediment conditions may be mutagenic •these genes may correct DNA defects resulting from sediment mutagens (e.g., organic acid fermentation products)

  20. Response to mutagens • One mutant B12(pF11) belongs to UmuC family. • It has 53.83% similarity to D. vulgarisumuC protein.

  21. Protection from a Severe Mutagenic Event The SOS response Cited from: Genetics 148: 1599–1610 (April, 1998)

  22. STM mutants in MR-1 with potential role in drug efflux COG Cation/Multidrug efflux pump Multidrug resistance efflux pump Multidrug resistance efflux pump Transcriptional regulator

  23. mexF deletion cannot grow in the presence of 5 g/ml chloramphenicol WT MR-1 & ∆mexF w/o antibiotic WT MR-1 + antibiotic ∆mexF

  24. Tripartite efflux pump system antibiotic Outer membrane protein Outer membrane Periplasmic space Membrane fusion protein (MexE) Inner membrane RND Exporter (MexF) Adapted from Aeschlimann, 2004 RND=resistance-nodulation-division

  25. Other environmental bacteria proteins similar to MR-1 MexF from NCBI pblast; Gaps range from 0-3% -proteobacteria -proteobacteria Other /-proteobacteria -proteobacteria

  26. What have we learned from STM • Identified a number of genes and cell functions that are required for life in the real world but not needed for growth in culture. • Demonstrated for the first time the importance of specific genes as cells are growing in natural environments. • Raised many questions regarding function of specific genes. Regulatory genes. Protection from toxins. DNA repair, etc.

  27. Acknowledgments • Funding from DOE ERSP program. • Qingwei Luo, Jennifer Groh, Xiangkai Li and Nydia Castaneda. • EMSL

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