MOLECULAR MECHANISMS OF MICROBIAL SULFUR AND SELENIUM REDUCTION

Sulfur and selenium are redox-active elements whose speciation is governed largely by microbial reduction. For sulfur, redox state determines chemical reactivity; for selenium, it determines mobility and toxicity. Anaerobic bacteria carry out these reductions through modular respiratory pathways that deliver electrons to a wide range of terminal acceptors, yet for many sulfur and selenium compounds the pathways and enzymes responsible remain undefined, and it is unclear how readily these components function outside the organisms that encode them. The objective of this dissertation was to define the molecular mechanisms by which bacteria reduce sulfur and selenium compounds under anaerobic conditions and, for selenate reduction, to determine how readily those mechanisms transfer between species. Chapter 2 examined the extracellular reduction of organic disulfides by Shewanella oneidensis MR-1, a respiratory generalist whose interactions with organic sulfur have remained poorly understood. Using the membrane-impermeant model disulfide 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), a transposon mutagenesis screen identified a mutant that had lost approximately 90% of its DTNB reduction activity through disruption of dsbD, which encodes the oxidoreductase required for cytochrome c maturation. Because DsbD is required to assemble the multiheme c-type cytochromes of the extracellular electron transfer pathway, that pathway was tested directly, and mutants lacking Mtr proteins were severely impaired in disulfide reduction. These results establish that S. oneidensis reduces extracellular organic disulfides through the same Mtr pathway it uses to respire metal oxides, extending the substrate range of extracellular electron transfer to a defined organic functional group. Chapter 3 identified the genetic basis of dissimilatory selenate reduction in the facultative anaerobe Enterobacter cloacae SLD1a-1, characterized biochemically for decades yet whose selenate reductase gene had remained unidentified. Genome sequencing revealed a membrane-bound, twin-arginine translocation (TAT)-exported molybdoenzyme operon, designated srnABCD and regulated by the fumarate and nitrate reductase regulator (FNR). Mutagenesis of the catalytic subunit gene srnA abolished selenate reduction, and complementation restored it. Heterologous expression of srnA in Escherichia coli, which does not natively reduce selenate, conferred activity, establishing that the reductase remains functional outside its native host. Chapter 4 extended this question to S. oneidensis MR-1, which cannot natively reduce selenate, the most oxidized and mobile selenium oxyanion. The srnABCD selenate reductase operon was introduced into MR-1 to generate a strain, designated TVN, and to test whether it remains functional within the distinct electron transfer network of this organism. Together, these studies define the pathway responsible for extracellular disulfide reduction and the gene responsible for dissimilatory selenate reduction, and demonstrate that the genetic determinants of selenate reduction remain functional outside their native host, with implications for the fate and bioremediation of selenium in contaminated environments.