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Microbiology of arsenic redox transformations

Environment. Molecular Genetics. Arsenic. Public Health. Microbiology of arsenic redox transformations. UCSC. Chad Saltikov and collaborators. Arsenic Contamination. Arsenite Oxidizer. As(III). Arsenate Reducing Bacterium. Microbial redox transformations of arsenic. Aqueous.

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Microbiology of arsenic redox transformations

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  1. Environment Molecular Genetics Arsenic Public Health Microbiology of arsenic redox transformations UCSC Chad Saltikov and collaborators

  2. Arsenic Contamination Arsenite Oxidizer As(III) Arsenate Reducing Bacterium Microbial redox transformations of arsenic Aqueous Sediment

  3. Phylogenetic relationship of arsenic metabolizing microbes

  4. Arsenic oxidation and reduction pathways Arsenite Oxidation Arsenate Respiration Arsenic Detoxification Adapted from Silver and Phung 2005. Appl. Env. Micro. 71(2):599-608

  5. Oxidases and reductases are in a large family of proteins

  6. arrA genes from other bacteria Akaliphilus metalliredigenes: 66%* Geobacter uraniumreducens: 65%* Desulfosporosinus sp. Y5: 66%* *aa similarities to ArrA of ANA-3

  7. The arsenic island of Shewanella:ANA-3, CN-32, and W3-18-1 CN-32 W3-18-1

  8. What environmental conditions trigger arsenate reduction?ArsC (detoxify)vs.ArrA (respire) As(V) As(III)

  9. Monitor two arsenate reduction pathwaysin our model arsenate reducer Shewanella sp. ANA-3 (an)aerobic vs. As Monitor the transcription of arrA and arsC Saltikov and Newman 2003 PNAS 100(19):10983-10988

  10. Quantify gene specific mRNA: Grow culture to mid log Extract/Purify RNA Reverse Transcribe RNA Quantify gene- specific mRNA by real time PCR 0.04 ng

  11. Dynamic expression in various growth phases with As(V) as electron acceptor Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

  12. How does phosphate influence As(V) respiration and arr/ars expression As(V)+Pi Fumarate+Pi Expression Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

  13. What about other electron acceptors? As(III) = inducer O2 and NO3- inhibit Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

  14. No As No As What are the sensitivities of arrA and arsC expression to As? A. B. Wild-type ∆arrA, ∆arsC Log [arsenite] µM Log [arsenate] µM Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

  15. Are cytochromes required for arsenate respiration in our model organism?factoid: Shewanella has 39 reading frames encoding c-type cytochromesIron containing proteins similar to heme of a red blood cell.

  16. CymA--tetraheme cytochromeis required for respiring arsenate periplasm As(V) As(III) ArrAB UQH2 UQ dehydrogenase cytoplasm Adapted from Schwalb et al. 2003 Biochemistry 42(31):9491-9497

  17. cymA is required for respiring As(V) in CN-32 Growth on nitrate Growth on arsenate

  18. cymA restores growth on As(V)

  19. Secondary structure prediction of CymA Inner Membrane

  20. Model for microbial arsenate reduction Respiration Detoxification

  21. Environmental significance of arsenate respiratory reduction

  22. A. ArrA protein and primer design L. Ladder: 100 bp ANA-3 arrA deleted Shew. oneidensis MR-1 Desulfitobacterium dehalogenens E. coli Pseudomonas chloraphis Shewanella sp. ANA-3 Desulfito. hafniense Desulfito. frappieri D. strain GBFH Wolinella sp. Wolinella succinogenes Citrobacter sp. Bacillus str. E1H Bacillus, str. MLS10 Sulfur. barnesii SES-3 Shewanella sp. HAR-4 Chrysiogenes OREX-4 Pyrobac. arsenaticum B. Detection in various strains arrA primers 16S rDNA primers 16S rDNA primers Malasarn et al. 2004 Science 06: 455 Can arrA be used to monitor and track As(V) reduction?

  23. * Pore water concentrationsof total As, Fe, and Mn inFe/As rich reservoir Mn Fe As Kneebone et al. 2002 ES&T 36(3):381-386

  24. 1 2 3 4 5 6 7 8 Blanks New primers for detecting arrA-like genes in As-enriched sediments Increasing depth in core 1517 1200 1000 500 400 300 Primer Dimmers 200 100

  25. Conclusions • Two genetic pathways for arsenate reduction • Respiratory by arr and detoxification by ars • Respiration pathway triggered by As: • As(III) > As(V) • Repressed by nitrate and oxygen • The mechanism for arsenate reduction involves other components in the cell. • The arrA gene is a useful marker for As redox • Gene copy number seems to correlate with redox gradients of As … more work to be done.

  26. Acknowledgments/Collaborators • UC Santa Cruz • Julie Nilsen • Caltech • Prof. Dianne Newman, Prof. Janet Hering, Rich Wildman • USGS • Dr. Ron Oremland, Dr. Thomas Kulp, Dr. Larry Miller, Shelly Hoeft

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