Isolation and characterization of multicytochrome gene cluster from Shewanella putrefaciens MR-1 involved in iron and manganese reduction

Dissimilatory Fe(III) and Mn(IV) reduction is an anaerobic respiratory process common to many environments. A large number of bacteria that utilize these metals as terminal electron acceptors have been isolated and identified for the past decade. However, the molecular mechanisms of metal reduction remain unclear despite intensive research in this area. There are no genes or proteins identified, which are directly involved in metal reduction. The major goal of this work is to identify and isolate genes related to Fe(III) and Mn(IV) reduction as well as to determine the specific functions of their products. The organism used in this work is the facultatively anaerobic metal reducer S. putrefaciens MR-1. Transposon mutagenesis was used to generate mutants solely deficient in Fe(III) and Mn(IV) reduction. Analysis of the interrupted regions in two such mutants, SR-8 and SR-21, led to the identification of three genes, designated mtrC, mtrA and mtrB. The deduced amino acid sequence of mtrC and mtrA revealed that these genes encode deca-heme c-type cytochromes. The third gene, mtrB, was shown to encode an outer membrane protein of 679 amino acids. mRNA and Western blot analyses indicated that the three genes are organized in a single operon, mtrCAB, which is expressed constitutively. To elucidate the role of each protein in metal reduction, gene replacement was used to generate strains lacking either mtrC, mtrA or mtrB. Whole-cell suspensions of mtrC−A+B+, mirC+A−B+ and mtrC+A+B− mutants resulted in 4.6-fold, 31-fold and 70-fold decrease in iron reduction rates, respectively. In contrast to whole cells, the levels of iron reductase activity in the crude cell extracts of the mutants were similar to that observed in the wild type. These findings strongly suggest that the products of mtrC, mtrA and mtrB play an important role in metal reduction, however, they may not constitute the terminal metal reductase complex. Analysis of the DNA region upstream of mtrCAB revealed a presence of another multicytochrome gene cluster. Although the identified genes, mtrD, mtrE, mtrF and omcA, exhibited high degree of similarity to the mtrCAB operon, there was no evidence indicating their involvement in Fe(III) and Mn(IV) reduction in S. putrefaciens