Dissimilative Fe(III) Reduction by Alteromonas putrefaciens Strain 200

Complementary genetic and biochemical analyses have been used to study the (dissimilative) iron reduction system of Alteromonas putrefaciens strain 200. Preliminary kinetic data suggested that A. putrefaciens 200 possessed a ferri-reductase that was physiologically distinct from either cytochrome oxidase or nitrate reductase. A suite of iron-reduction-deficient mutants was generated via chemical (EMS) and transposon (Tn5) mutagenesis procedures. A newly developed screening technique was subsequently used to identify mutants deficient in both high-rate and low-rate iron reduction activity. A conjugal gene transfer system was developed for mobilization of IncP1-based cloning vectors to A. putrefaciens 200. The broad host range (IncPl) cosmid cloning vector pVK100 was used to construct A. putrefaciens 200 gene clone banks in E. coli strains HB101 and S17-1 (mobilizing strain). Both three-way and two-way mating (conjugation) procedures were used to mobilize the gene clone banks into the suite of iron-reduction-deficient mutants during genetic (complementation) analysis. Two iron reduction clones (designated S4-E-2 and S18-F-4) were identified by their ability to restore iron reduction activity to several of the iron-reduction-deficient mutants. Preliminary biochemical characterization of selected mutant strains has indicated that cytochrome content may play an important role in the iron reduction process. Based on the results of the complementary genetic and biochemical studies, the iron reduction mutants have been placed into four classes: Class I (deficient in both high-rate and low-rate iron reduction activity, complemented by clones S4-E-2 and S18-F-4, possible b- or c-type cytochrome mutants), Class II (deficient in both high-rate and low-rate iron reduction activity, complemented by clone S4-E-2 but not by S18-F-4), Class III (proficient in high-rate iron reduction activity, but deficient in low-rate iron reduction activity, not complemented by either clone S4-E-2 or S18-F-4), and Class IV (deficient in both high-rate and low-rate iron reduction activity, but not complemented by either clone S4-E-2 or S18-F-4, possible d-type cytochrome or anaerobic regulatory mutant).