Shewanella oneidensis MR-1 Restores Menaquinone Synthesis to a Menaquinone-Negative Mutant

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.     

MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1

When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO2) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg2+, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.     

MtrB Is Required for Proper Incorporation of the Cytochromes OmcA and OmcB into the Outer Membrane of Shewanella putrefaciens MR-1

When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO₂) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg²⁺, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.     

Cell surface exposure of the outer membrane cytochromes of Shewanella oneidensis MR-1

AIM: To determine if the outer membrane (OM) cytochromes of the metal-reducing bacterium Shewanella oneidensis MR-1 are exposed on the cell surface. METHODS AND RESULTS: MR-1 cells were incubated with proteinase K or buffer and the resulting degradation of the OM cytochromes was examined by Western blotting. The periplasmic fumarate reductase (control) was not degraded. The OM cytochromes OmcA and OmcB were significantly degraded by proteinase K (71 and 31%, respectively). Immunofluorescence confirmed a prominent cell surface exposure of OmcA and a partial exposure of OmcB and the noncytochrome OM protein MtrB. CONCLUSIONS: The cytochromes OmcA and OmcB are exposed on the outer face of the OM. SIGNIFICANCE AND IMPACT OF THE STUDY: The cell surface exposure of these cytochromes could allow them to directly contact extracellular insoluble electron acceptors (e.g. manganese oxides) and is consistent with their in vivo role.     

The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination. METHODS AND RESULTS: In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled. The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes. Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA. The identity of this band was confirmed by its absence in an OmcA-minus mutant. A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method. CONCLUSIONS: Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed. SIGNIFICANCE: The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM. The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g. manganese oxides) and is consistent with its in vivo role.     

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.     

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.     

Elevated intracellular cyclic-di-GMP level in Shewanella oneidensis increases expression of c-type cytochromes

Electrochemically active biofilms are capable of exchanging electrons with solid electron acceptors and have many energy and environmental applications such as bioelectricity generation and environmental remediation. The performance of electrochemically active biofilms is usually dependent on c-type cytochromes, while biofilm development is controlled by a signal cascade mediated by the intracellular secondary messenger bis-(3ʹ-5ʹ) cyclic dimeric guanosine monophosphate (c-di-GMP). However, it is unclear whether there are any links between the c-di-GMP regulatory system and the expression of c-type cytochromes. In this study, we constructed a S. oneidensis MR-1 strain with a higher cytoplasmic c-di-GMP level by constitutively expressing a c-di-GMP synthase and it exhibited expected c-di-GMP-influenced traits, such as lowered motility and increased biofilm formation. Compared to MR-1 wild-type strain, the high c-di-GMP strain had a higher Fe(III) reduction rate (21.58 vs 11.88 pM of Fe(III)/h cell) and greater expression of genes that code for the proteins involved in the Mtr pathway, including CymA, MtrA, MtrB, MtrC and OmcA. Furthermore, single-cell Raman microspectroscopy (SCRM) revealed a great increase of c-type cytochromes in the high c-di-GMP strain as compared to MR-1 wild-type strain. Our results reveal for the first time that the c-di-GMP regulation system indirectly or directly positively regulates the expression of cytochromes involved in the extracellular electron transport (EET) in S. oneidensis, which would help to understand the regulatory mechanism of c-di-GMP on electricity production in bacteria.     

Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli as ferric reductases

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli , we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c -type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10× and 30× higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.     

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.     

Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

Vanadium(V) reduction by Shewanella oneidensis MR-1 requires menaquinone and cytochromes from the cytoplasmic and outer membranes

The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

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.     

Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

Vanadium(V) Reduction by Shewanella oneidensis MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes

The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.     

Characterization of Protein-Protein Interactions Involved in Iron Reduction by Shewanella oneidensis MR-1

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.     

Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.