OmcB,a c-type polyheme cytochrome,involved in Fe(III) reduction in Geobacter sulfurreducens

Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.     

MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens

A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).     

Evidence that OmcB and OmpB of Geobacter sulfurreducens are outer membrane surface proteins

The c-type cytochrome (OmcB) and the multicopper protein (OmpB) required for Fe(III) oxide reduction by Geobacter sulfurreducens were predicted previously to be outer membrane proteins, but it is not clear whether they are positioned in a manner that permits the interaction with Fe(III). Treatment of whole cells with proteinase K inhibited Fe(III) reduction, but had no impact on the inner membrane-associated fumarate reduction. OmcB was digested by protease, resulting in a smaller peptide. However, immunogold labeling coupled with transmission electron microscopy did not detect OmcB, suggesting that it is only partially exposed on the cell surface. In contrast, OmpB was completely digested with protease. OmpB was loosely associated with the cell surface as a substantial portion of it was recovered in the culture supernatant. Immunogold labeling demonstrated that OmpB associated with the cell was evenly distributed on the cell surface rather than localized to one side of the cell like the conductive pili. Although several proteins required for Fe(III) oxide reduction are shown to be exposed on the outer surface of G. sulfurreducens, the finding that OmcB is also surface exposed is the first report of a protein required for optimal Fe(III) citrate reduction at least partially accessible on the cell surface.     

A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB

The Gram-negative bacterium Shewanella oneidensis MR-1 shows a remarkably versatile anaerobic respiratory metabolism. One of its hallmarks is its ability to grow and survive through the reduction of metallic compounds. Among other proteins, outer membrane decaheme cytochromes c OmcA and OmcB have been identified as key players in metal reduction. In fact, both of these cytochromes have been proposed to be terminal Fe(III) and Mn(IV) reductases, although their role in the reduction of other metals is less well understood. To obtain more insight into this, we constructed and analyzed omcA, omcB and omcA/omcB insertion mutants of S. oneidensis MR-1. Anaerobic growth on Fe(III), V(V), Se(VI) and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III) reduction, a redundant function in V(V) reduction, and no apparent involvement in Se(VI) and U(VI) reduction. Growth of the omcB(-) mutant on Fe(III) was more affected than growth of the omcA(-) mutant, suggesting OmcB to be the principal Fe(III) reductase. This result was corroborated through the examination of whole cell kinetics of OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is approximately 11.5 and approximately 6.3 times faster than OmcA at saturating and low nonsaturating concentrations of Fe(III)-nitrilotriacetic acid, respectively, whereas the omcA(-) omcB(-) double mutant was devoid of Fe(III)-nitrilotriacetic acid reduction activity. These experiments reveal, for the first time, that OmcA and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis MR-1. Kinetic inhibition experiments further revealed vanadate (V(2)O(5)) to be a competitive and mixed-type inhibitor of OmcA and OmcB, respectively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Neither sodium selenate nor uranyl acetate were found to inhibit OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken together with our growth experiments, this suggests that proteins other than OmcA and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.     

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.     

The periplasmic 9.6-kilodalton c-type cytochrome of Geobacter sulfurreducens is not an electron shuttle to Fe(III)

Geobacter sulfurreducens contains a 9.6-kDa c-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions of G. sulfurreducens it did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellular c-type cytochromes in cultures of G. sulfurreducens demonstrated that the dominant c-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.     

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile

Background

In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.

Results

The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).

Conclusion

This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.     

PilR, a transcriptional regulator for pilin and other genes required for Fe(III) reduction in Geobacter sulfurreducens

Growth using Fe(III) as a terminal electron acceptor is a critical physiological process in Geobacter sulfurreducens. However, the mechanisms of electron transfer during Fe(III) reduction are only now being understood. It has been demonstrated that the pili in G. sulfurreducens function as microbial nanowires conducting electrons onto Fe(III) oxides. A number of c-type cytochromes have also been shown to play important roles in Fe(III) reduction. However, the regulatory networks controlling the expression of the genes involved in such processes are not well known. Here we report that the expression of pilA, which encodes the pilistructural protein, is directly regulated by a two-component regulatory system in which PilR functions as an RpoN-dependent enhancer binding protein. Surprisingly, a deletion of the pilR gene affected not only insoluble Fe(III) reduction, which requires pili, but also soluble Fe(III) reduction, which, in contrast, does not require pili. Gene expression profiling using whole-genome DNA microarray and quantitative RT-PCR analyses obtained with a PilR-deficient mutant revealed that the expression of pilA and other pilin-related genes are downregulated, while many c-type cytochromes involved in Fe(III) reduction were differentially regulated. This is the first instance of an enhancer binding protein implicated in regulating genes involved in Fe(III) respiratory functions.     

Overlapping role of the outer membrane cytochromes of Shewanella oneidensis MR-1 in the reduction of manganese(IV) oxide

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 have distinct or overlapping roles in the reduction of insoluble manganese(IV) oxide. METHODS AND RESULTS: The gene replacement mutant (OMCA1) which lacks OmcA was partially deficient in Mn(IV) reduction. Complementation of OMCA1 with a vector (pVK21) that contains omcB but not omcA restored Mn(IV) reduction to levels that were even greater than those of wild-type. Examination of the OM of OMCA1/pVK21 revealed greater than wild-type levels of OmcB protein and specific haem content. CONCLUSIONS: Overexpression of OmcB can compensate for the absence of OmcA in the reduction of insoluble Mn(IV) oxides. Therefore, there is at least a partial overlap in the roles of these OM cytochromes in the reduction of insoluble Mn(IV) oxide. SIGNIFICANCE: The overlapping roles of these two cytochromes has important implications for understanding the mechanism by which MR-1 reduces insoluble metal oxides. There is no obligatory sequential electron transfer from one cytochrome to the other. They could both potentially serve as terminal reductases for extracellular electron acceptors.     

Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.