1. Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC

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

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 W3-18-1 CN-32

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

9. Monitor two arsenate reduction pathways in 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)+PiFumarate+PiExpression Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

13. What about other electron acceptors? As(III) = inducer O 2 and NO 3 - inhibit Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396

14. What are the sensitivities of arrA and arsC expression to As? Log [arsenite] µMLog [arsenate] µM ∆arrA, ∆arsCWild-type A.B. No As No As 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 cytochromes Iron containing proteins similar to heme of a red blood cell.

16. CymA--tetraheme cytochrome is required for respiring arsenate UQH 2 UQ periplasm cytoplasm dehydrogenase ArrAB As(V) As(III) Adapted from Schwalb et al. 2003 Biochemistry 42(31):9491-9497

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

18. cymA restores growth on As(V)

19. Secondary structure prediction of CymA Inner Membrane

20. Model for microbial arsenate reduction Detoxification Respiration

21. Environmental significance of arsenate respiratory reduction

22. Can arrA be used to monitor and track As(V) reduction? A. ArrA protein and primer design L. Ladder: 100 bp 1.ANA-3 arrA deleted 2.Shew. oneidensis MR-1 3.Desulfitobacterium dehalogenens 4.E. coli 5.Pseudomonas chloraphis 6.Shewanella sp. ANA-3 7.Desulfito. hafniense 8.Desulfito. frappieri 9.D. strain GBFH 10.Wolinella sp. 11.Wolinella succinogenes 12.Citrobacter sp. 13.Bacillus str. E1H 14.Bacillus, str. MLS10 15.Sulfur. barnesii SES-3 16.Shewanella sp. HAR-4 17.Chrysiogenes 18.OREX-4 19.Pyrobac. arsenaticum 16S rDNA primers arrA primers 16S rDNA primers B. Detection in various strains Malasarn et al. 2004 Science 06: 455

23. Pore water concentrations of total As, Fe, and Mn in Fe/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 1517 1200 1000 500 400 300 200 100 New primers for detecting arrA-like genes in As-enriched sediments Increasing depth in core Primer Dimmers

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