The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).     

The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis

The tetraheme c-type cytochrome, CymA, from Shewanella oneidensis MR-1 has previously been shown to be required for respiration with Fe(III), nitrate, and fumarate [Myers, C. R., and Myers, J. M. (1997) J. Bacteriol. 179, 1143-1152]. It is located in the cytoplasmic membrane where the bulk of the protein is exposed to the periplasm, enabling it to transfer electrons to a series of redox partners. We have expressed and purified a soluble derivative of CymA (CymA(sol)) that lacks the N-terminal membrane anchor. We show here, by direct measurements of electron transfer between the purified proteins, that CymA(sol) efficiently reduces S. oneidensis fumarate reductase. This indicates that no further proteins are required for electron transfer between the quinone pool and fumarate if we assume direct reduction of CymA by quinols. By expressing CymA(sol) in a mutant lacking CymA, we have shown that this soluble form of the protein can complement the defect in fumarate respiration. We also demonstrate that CymA is essential for growth with DMSO (dimethyl sulfoxide) and for reduction of nitrite, implicating CymA in at least five different electron transfer pathways in Shewanella.     

Electrochemical interrogations of the Mtr cytochromes from <Emphasis Type="Italic">Shewanella</Emphasis>: opening a potential window

The multi-heme cytochromes from Shewanella oneidensis associated with the dissimilatory metal reduction (DMR) pathway have been investigated using the technique of protein film voltammetry (PFV). Using PFV, we have interrogated each of the multi-heme cytochromes (MtrA, STC, and solubilized versions of the membrane-bound proteins CymA, OmcA, and MtrC) under identical conditions for the first time. Each cytochrome reveals a broad envelope of voltammetric response, indicative of multiple redox cofactors that span a range of potential of approximately 300 mV. Our studies show that, when considered as an aggregate pathway, the multiple hemes of the DMR cytochromes provide a "window" of operating potential for electron transfer to occur from the cellular interior to the exterior spanning values of -250 to 0 mV (at circumneutral values of pH). Similarly, each cytochrome supports interfacial electron transfer at rates on the order of 200 s(-1). These data are taken together to suggest a model of electron transport where a wide window of potential allows for charge transfer from the cellular interior to the exterior to support bioenergetics.     

Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway

Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye‐containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR‐1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox‐active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.     

Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1

In this work, we investigated the anaerobic decolorization of methyl orange (MO), a typical azo dye, by Shewanella oneidensis MR-1, which can use various organic and inorganic substances as its electron acceptor in natural and engineered environments. S. oneidensis MR-1 was found to be able to obtain energy for growth through anaerobic respiration accompanied with dissimilatory azo-reduction of MO. Chemical analysis shows that MO reduction occurred via the cleavage of azo bond. Block of Mtr respiratory pathway, a transmembrane electron transport chain, resulted in a reduction of decolorization rate by 80%, compared to the wild type. Knockout of cymA resulted in a substantial loss of its azo-reduction ability, indicating that CymA is a key c-type cytochrome in the electron transfer chain to MO. Thus, the MtrA-MtrB-MtrC respiratory pathway is proposed to be mainly responsible for the anaerobic decolorization of azo dyes such as MO by S. oneidensis.     

Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates

Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells.     

Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR‐1

Four distinct pathways predicted to facilitate electron flow for respiration of externally located substrates are encoded in the genome of Shewanella oneidensis strain MR‐1. Although the pathways share a suite of similar proteins, the activity of only two of these pathways has been described. Respiration of extracellular substrates requires a mechanism to facilitate electron transfer from the quinone pool in the cytoplasmic membrane to terminal reductase enzymes located on the outer leaflet of the outer membrane. The four pathways share MtrA paralogues, a periplasmic electron carrier cytochrome, and terminal reductases similar to MtrC for reduction of metals, flavins and electrodes or to DmsAB for reduction of dimethyl sulphoxide (DMSO). The promiscuity of respiratory electron transfer reactions catalysed by these pathways has made studying strains lacking single proteins difficult. Here, we present a comprehensive analysis of MtrA and MtrC paralogues in S. oneidensis to define the roles of these proteins in respiration of insoluble iron oxide, soluble iron citrate, flavins and DMSO. We present evidence that some periplasmic electron carrier components and terminal reductases in these pathways can provide partial compensation in the absence of the primary component, a phenomenon described as modularity, and discuss biochemical and evolutionary implications.     

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.     

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.     

Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the beta-barrel protein MtrB

The Shewanella oneidensis outer membrane β-barrel protein MtrB is part of a membrane-spanning protein complex (MtrABC) which is necessary for dissimilatory iron reduction. Quantitative PCR, heterologous gene expression, and mutant studies indicated that MtrA is required for periplasmic stability of MtrB. DegP depletion compensated for this MtrA dependence.     

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.     

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.     

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately -240 mV) and low-spin (approximately -110, -190 and -265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E(m) = -80 mV) in the presence of NADH (E(m) = -320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.     

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.     

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.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.