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The Role of Small Molecules in Anaerobic Respiration of Shewanella Jeffrey Gralnick Assistant Professor University of Minnesota Department of Microbiology BioTechnology Institute [email protected] Respiratory Diversity • • • • • • • Oxygen Nitrate Nitrite TMAO DMSO Sulfur Fumarate • • • • • • • • • • Chromium Selenium Arsenic Technetium Uranium Tellurium Cobalt Vanadium Manganese Iron Respiration of insoluble substrates Iron Oxide Manganese Oxide “Extracellular Respiration” Why is this important? • Redox state of metals almost always influences their solubility, toxicity or both. • Respiration of insoluble substrates requires electron transfer pathways that E. coli has never known. • The ability to transfer electrons to the outside of the cell allows us to directly harvest respiratory energy in microbial fuel cells. Model for iron oxide respiration in S. oneidensis OmcA MtrC OmcA MtrB MtrA CymA MQ Iron Oxide Reduction in S. oneidensis omcA mtrA mtrC mtrB The molecular mechanism is now well understood at the genetic level. What is happening between the cell and the mineral? ? Methods of extracellular respiration to iron oxide Gralnick and Newman, 2007 Shewanella can reduce substrates they can’t physically touch. Lies et al., 2005 Respiration of electrodes by Shewanella OmcA MtrC OmcA MtrB MtrA CymA MQ Current ( A) Monitoring electron flow from S. oneidensis over time Time (hours) Baron, Bond A soluble factor is contributing to electricity generation. Marsili et al., 2008 Riboflavin was the primary constituent as determined by LC/MS/MS von Canstein et al., 2008 B2 - Riboflavin ~ 250 nM Marsili et al., 2008 Riboflavin is a redox active vitamin - can Shewanella directly reduce it? Relative Fluorescence Units Riboflavin reduction assay No electron donor With lactate Time (seconds) B2 Reduction Rate* Riboflavin reduction rates B2(ox) MR-1 omcA mtrC B2(red) mtrC mtrB mtrA omcA * Fluor / CFU / second B2 appears to be reduced primarily on the outside of the cell. How are flavins exported out of the cell and do flavin concentrations correlate with rates of iron oxide reduction? Total Flavins (M) Mutants isolated that accumulate less external flavin MR-1 JG412 JG413 Early (7 hours) Late (70 hours) Neither mutant was completely defective in external flavin accumulation JG412 : Predicted sensor kinase JG413 : Predicted phosphatase Mutants that accumulate less external flavin reduce iron oxide slowly mM Fe(II) MR-1 JG413 JG412 Flavin accumulation correlates with the strain’s ability to reduce iron oxide Can we engineer a strain that accumulates less external flavin? • Riboflavin biosynthetic gene clusters in S. oneidensis: ribD ribE-2 ribBA ribH SO2295 ribE-1 • FMN riboswitches control gene expression of flavin biosynthesis in E. coli and B. subtilis. • Over-expression of riboflavin kinase should increase internal FMN levels, potentially decreasing expression of flavin biosynthetic genes. ribE-2 pBBR1MCS-2 External flavin accumulation is increased in the ribE-2 overexpression strain Riboflavin Flavin (M) FMN ~ 3 fold increase in external flavin levels observed. This result is inconsistent with the existence of an FMN riboswitch to repress flavin biosynthesis. mM Fe(II) Flavin over-producing strain reduces iron oxide faster than wild-type Flavin accumulation correlates with the strain’s ability to reduce iron oxide