A genetic system for studying uranium reduction by Shewanella putrefaciens

A U(VI) reduction-deficient mutant (Urr) screening technique was developed and combined with chemical mutagenesis procedures to identify several Urr mutants of Shewanella putrefaciens strain 200. The Urr mutants lacked the ability to grow anaerobically on U(VI) and various other compounds as electron acceptors. In particular, Urr mutant U14 lacked the ability to grow anaerobically on U(VI) and NO2-, yet retained the ability to grow anaerobically on eight other compounds as terminal electron acceptor. Several Fe(III) and Mn(IV) reduction-deficient mutants of S. putrefaciens 200 displayed Urr-positive phenotypes when tested for U(VI) reduction capability via the Urr mutant screening technique. This is the first reported isolation of a respiratory mutant that is unable to grow anaerobically on U(VI) as terminal electron acceptor. U(VI) reduction activity was restored in each mutant via conjugal mating experiments with a wild-type S. putrefaciens clone bank. In particular, the recombinant cosmid restoring U(VI) reduction activity to Urr mutant U14 was subcloned to 12-kb DNA fragment.