Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction

Abstract

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

FIG. 1

Levels of Fe(III) reduction in wild-type MR-1, mutant SR-21, and complemented mutant SR-21C. Reduction of Fe(III) to Fe(II) was monitored using ferrozine at 562 nm.

FIG. 2

Mn(IV) reduction by MR-1, SR-21, and SR-21C. Reduction of Mn(IV) is detected by clearing of brown color in the medium as seen in wild-type MR-1 and the complemented mutant SR-21C but not in the mutant SR-21.

FIG. 3

Reduction of fumarate, DMSO, TMAO, and nitrite by MR-1 and SR-21. Cell extracts of MR-1 and SR-21 were separated by native PAGE and assayed for fumarate (A), DMSO (B), and TMAO (C) reductase activities. Benzyl viologen was used as the artificial electron donor. Clear bands in all samples indicate the ability of MR-1 and SR-21 to reduce these electron acceptors. (D) Nitrite reduction by MR-1 and SR-21 was assayed by monitoring the decrease in nitrite concentration in the medium. No differences were observed in the ability of SR-21 to use nitrite compared to the wild type.

FIG. 4

Amino acid sequence alignment of NrfB from E. coli and the deduced amino acid sequence of MtrA. Identical amino acids are shaded and similar amino acids are boxed.

FIG. 5

SDS-PAGE of inner and outer membrane proteins of anaerobically grown MR-1, SR-21, and SR-21C. Lanes: 1 and 2, cell membrane fractions from MR-1 and SR-21, respectively; 3, 4, and 5, outer membrane fractions of MR-1, SR-21, and SR-21C, respectively. The band that corresponds to MtrB, which is not detected in the outer membrane fraction of the mutant SR-21, is indicated by an arrow. Numbers correspond to protein molecular mass markers (in kilodaltons).