ABSTRACTMicrobial iodate (IO3−) reduction is a major component of iodine biogeochemical cycling and is the basis of alternative strategies for remediation of iodine-contaminated environments. The molecular mechanism of microbial IO3− reduction, however, is not well understood. In several microorganisms displaying IO3− and nitrate (NO3−) reduction activities, NO3− reductase is postulated to reduce IO3− as alternate electron acceptor. In the present study, whole genome analyses of 25 NO3−-reducing Shewanella strains identified various combinations of genes encoding one assimilatory (cytoplasmic Nas) and three dissimilatory (membrane-associated Nar and periplasmic Napα and Napβ) NO3− reductases. Shewanella oneidensis was the only Shewanella strain whose genome encoded a single NO3− reductase (Napβ). Terminal electron acceptor competition experiments in S. oneidensis batch cultures amended with both NO3− and IO3− demonstrated that neither NO3− nor IO3− reduction activities were competitively inhibited by the presence of the competing electron acceptor. The lack of involvement of S. oneidensis Napβ in IO3− reduction was confirmed via phenotypic analysis of an in-frame gene deletion mutant lacking napβA (encoding the NO3−-reducing NapβA catalytic subunit). S. oneidensis ΔnapβA was unable to reduce NO3−, yet reduced IO3− at rates higher than the wild-type strain. Thus, NapβA is required for dissimilatory NO3− reduction by S. oneidensis, while neither the assimilatory (Nas) nor dissimilatory (Napα, Napβ, and Nar) NO3− reductases are required for IO3− reduction. These findings provide the first genetic evidence that IO3− reduction by S. oneidensis does not involve nitrate reductase and indicate that S. oneidensis reduces IO3− via an as yet undiscovered enzymatic mechanism.