The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate, and fumarate by Shewanella oneidensis MR-1

Abstract

Background: Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.

Results: Omp35 was purified from the OM of anaerobically grown cells, the gene encoding Omp35 was identified, and an omp35 null mutant (OMP35-1) was isolated and characterized. Although OMP35-1 grew on all electron acceptors tested, a significant lag was seen when grown on fumarate, nitrate, and Fe(III). Complementation studies confirmed that the phenotype of OMP35-1 was due to the loss of Omp35. Despite its requirement for wild-type rates of electron acceptor use, analysis of Omp35 protein and predicted sequence did not identify any electron transport moieties or predicted motifs. OMP35-1 had normal levels and distribution of known electron transport components including quinones, cytochromes, and fumarate reductase. Omp35 is related to putative porins from MR-1 and S. frigidimarina as well as to the PorA porin from Neisseria meningitidis. Subcellular fraction analysis confirmed that Omp35 is an OM protein. The seven-fold anaerobic upregulation of Omp35 is mediated post-transcriptionally.

Conclusion: Omp35 is a putative porin in the OM of MR-1 that is markedly upregulated anaerobically by a post-transcriptional mechanism. Omp35 is required for normal rates of growth on Fe(III), fumarate, and nitrate, but its absence has no effect on the use of other electron acceptors. Omp35 does not contain obvious electron transport moieties, and its absence does not alter the amounts or distribution of other known electron transport components including quinones and cytochromes. The effects of Omp35 on anaerobic electron acceptor use are therefore likely indirect. The results demonstrate the ability of non-electron transport proteins to influence anaerobic respiratory phenotypes.

Figure 1

Colony PCR reactions with primers specific for the omp35 gene or the chloramphenicol acetyltransferase gene (cat) from pEP185.2. Lanes 1 and 2 are reactions with omp35 primers O1 and O2 (see Table 2) and lanes 3–5 are reactions with cat primers C1 and C2 (see Table 2). The templates for PCR were as follows: lanes 1 and 3, MR-1; lanes 2 and 4, OMP35-1; lane 5, pDSEPomp35. The sizes of the DNA markers (lane M) in kilobases are indicated on the left.

Figure 2

Western blot of subcellular fractions of MR-1 and OMP35-1 with an antibody specific for Omp35. The lanes were loaded with 20 ng protein from each subcellular fraction; cytoplasmic membrane (CM), intermediate membrane (IM), outer membrane (OM), and soluble fraction (soluble). Fractions were isolated in duplicate from cells grown anaerobically with fumarate as the electron acceptor. The results shown are representative.

Figure 3

Relative levels of Omp35 protein (A, B) and omp35 transcript (C, D) in aerobically-grown versus fumarate-grown MR-1. A, B: Omp35 protein was detected by western blot of whole cells using an antibody specific for Omp35. An example of two dilutions of a representative experiment are shown in panel A, and the quantitative results from densitometric analysis of western blots from three independent experiments (mean ± S.D.) are shown in panel B. *, statistically different from aerobic to P ≤ 0.001. C, D: Transcript was determined by RNase protection using an antisense probe specific for the omp35 transcript. The data for three independent experiments are shown in panel C, and the quantitative results from densitometric analysis of the RPA blots (mean ± S.D.) are shown in panel D. For each experiment, dilutions of each sample were analyzed to ensure linearity of signal intensity.

Figure 4

Western blot of lysed whole cells with an antibody specific for Omp35. Each lane was loaded with equivalent wet cell pellet weight (30 μg). The strains carrying the various plasmids are indicated above each lane. This blot is representative of duplicate experiments. The bars and numbers at the right indicate the migration and mass of the protein standards. The results shown are for aerobically grown cells. Analogous results were obtained with fumarate-grown cells (not shown).

Figure 5

Anaerobic growth of various strains on fumarate (A) and nitrate (B). Values represent mean ± high/low for two parallel but independent experiments for each strain.

Figure 6

Anaerobic reduction (A) and growth (B) on Fe(III) citrate by various strains. One representative experiment from two independent experiments is shown.

Figure 7

Anaerobic reduction of αFeOOH by various strains. Values represent mean ± high/low for two parallel but independent experiments for each strain.

Figure 8

The effect of proteinase K on MR-1 proteins OmcA, fumarate reductase (FR), and Omp35. Either fumarate-grown whole cells (WC) or purified outer membrane (OM) fractions isolated from fumarate-grown MR-1 were treated with proteinase K. Results of duplicate experiments are expressed as the ratio of band intensity with proteinase K vs. protease-free control. *, statistically significant from FR control to P ≤ 0.006.

Figure 9

Amino acid sequence similarities between Omp35 of MR-1 and the top matches as identified by BLAST. Alignments were done using the ClustalW function of MacVector software. Identical residues are indicated by uppercase letters, analogous residues by lowercase letters, and unmatched residues by dots. Alignments were facilitated by introducing gaps (-). The numbers on the right indicate the relative numbering of residues within each immature protein; the total number of residues in each protein is shown in parentheses at the end of each sequence. Comparative sequences and their accession numbers are as follows: PorA N men (Neisseria meningitidis PorA, OM porin precursor, GenBank AF226349_1); SO1557 MR-1 (S. oneidensis MR-1, putative OM porin, TIGR genome locus SO1557); SO1420 MR-1 (S. oneidensis MR-1, putative OM porin, TIGR genome locus SO1420); IfcO S fri (S. frigidimarina, putative OM porin, GenBank AJ236923).