Sulphite respiration in W. succinogenes
It has been demonstrated here for the first time that W. succinogenes cells are able to grow by anaerobic sulphite respiration. This capacity adds to other dissimilatory reactions of W. succinogenes using sulphur compounds, namely polysulphide respiration with formate or hydrogen gas as electron donor and fumarate respiration using sulphide as electron donor (Macy et al., 1986; Schumacher et al., 1992; Klimmek et al., 2004). In contrast to Campylobacter jejuni, W. succinogenes was not reported to grow by microaerobic respiration using sulphite as electron donor and the corresponding sulphite:cytochrome c oxidoreductase (sor) genes are absent from its genome (Myers and Kelly, 2005).
Most likely, the octahaem cytochrome c MccA functions as the sole terminal reductase of sulphite respiration (see the corresponding model in Fig. 4). MccA was detected in sulphite-grown W. succinogenes wild-type cells for the first time since the protein is absent from cells grown with alternative electron acceptors indicating tight regulation of mccABCD-ccsA1 expression. This finding argues against a role of MccA in sulphite detoxification. The nature of the assumed sulphite-sensor and the corresponding signal transduction mechanism has not been investigated as yet. In this context it is notable that a two-component regulatory system named MccR/MccS is encoded immediately upstream of the mccABCD-ccsA1 gene cluster that might be involved in the synthesis of MccA (Fig. 4) (Hartshorne et al., 2006). The mccRS genes were only found in the genomes of W. succinogenes, Sulfurospirillum deleyianum and some, but not all, Campylobacter species (Fig. S4), whereas all other MccA-encoding bacteria including many Epsilonproteobacteria of the genus Campylobacter lack these genes.
Figure 4. Model of the electron transport chain catalysing electrogenic formate oxidation by sulphite in W. succinogenes. The left part shows the putative electron transport chain that catalyses the six-electron reaction of sulphite reduction by formate and comprises the electrogenic heterotrimeric formate dehydrogenase complex (FdhABC), the quinone (Q)/quinol (QH2) pool, the MccCD quinol dehydrogenase and MccA (see text for further details). Formate and sulphite are drawn in their protonated forms to illustrate the bioenergetics of sulphite respiration based on the coupled turnover of protons and electrons (underlined). While formate-dependent quinone reduction is electrogenic, quinol oxidation by sulphite is assumed to be electroneutral. The right part depicts the distinct functions of two cytochrome c haem lyases (CcsA1 and CcsA2) in periplasmic MccA maturation. Both haem lyases contain ten transmembrane segments and are assumed to transport haem b across the membrane (Kern et al., 2010b). The W. succinogenes mcc gene cluster is shown at the bottom. Mo, molybdenum cofactor; R, mccR gene.
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MccA represents a novel sulphide-generating cytochrome c sulphite reductase distinct from functionally similar sirohaem-containing enzymes. Isolated W. succinogenes MccA was shown previously to be inactive towards reduction of fumarate, nitrate, nitrite, thiosulphate, tetrathionate and Fe(III) complexes (Hartshorne et al., 2007). Therefore, MccA appears to be more specific than NrfA, which reduces nitrite and sulphite as well as nitric oxide, hydroxylamine and hydrogen peroxide (Lukat et al., 2008; Kern et al., 2011; Simon et al., 2011). Like MccA, NrfA also contains a special haem c group as the active site haem c is bound by a CX2CK haem c binding motif that donates a proximal lysine ligand to the haem c iron (Einsle et al., 2000; Pisa et al., 2002).
It was shown previously that MccA forms a periplasmic homotrimer under physiological conditions that contains 24 haem c groups (Hartshorne et al., 2007). One of the eight haem c groups of the MccA monomer was clearly distinct in showing a red-shifted absorption maximum in its reduced form. Furthermore, spectropotentiometric titration of MccA revealed that this haem c had a considerably more positive midpoint potential (+ 17 mV) than any other MccA haem c group (Hartshorne et al., 2007). These features are indicative of a special haem c group that is possibly involved in sulphite reduction and it seems to be an attractive idea that this haem c group is the one that is attached to the special CX15CH haem c binding motif (Fig. 4). On the other hand, previous EPR and UV/Vis spectroscopy did not suggest the presence of a high-spin haem c, which, however, might have been masked by strong electronic coupling (Hartshorne et al., 2007). The maturation of MccA is a complex process that involves the Sec system for apo-MccA secretion, the cytochrome c biogenesis system II (involving the distinct cytochrome c haem synthases CcsA2 and CcsA1) and probably also the assumed peptidyl-prolyl cis-trans isomerase MccB (Fig. 4) (Hartshorne et al., 2006; 2007; Kern et al., 2010a,b; Simon and Hederstedt, 2011).
Which components are involved in the set-up of a pmf generating electron transport chain catalysing formate oxidation by sulphite? In our current model (Fig. 4), the well-known electrogenic heterotrimeric formate dehydrogenase complex (FdhABC) couples formate oxidation to quinone reduction (Simon et al., 2008; Kern and Simon, 2009a). The quinol-reducing part of the electron transport chain is less clear but might minimally involve the putative quinol dehydrogenase module MccCD and MccA as the terminal reductase. MccD, a PsrC/NrfD homologue with eight putative membrane-spanning segments, might catalyse quinol oxidation and electron transfer to the iron-sulphur protein MccC, which is assumed to work as a specific redox mediator between MccD and MccA (Simon and Kern, 2008). It appears that MccC and MccD can not be functionally replaced by other proteins in W. succinogenes sulphite respiration under the tested conditions. At present, it is unknown which type of quinone is involved in W. succinogenes sulphite respiration. W. succinogenes is known to contain menaquinone6 as an essential quinone under fumarate- and nitrate-respiring conditions but produces methyl-menaquinone6 during polysulphide respiration (Dietrich and Klimmek, 2002). Methyl-menaquinone6 was shown to be involved in electron transport from formate to polysulphide and was proposed to bind to the membrane anchor subunit PsrC (a MccD homologue) of the polysulphide reductase complex PsrABC (Dietrich and Klimmek, 2002; Klimmek et al., 2004). This finding is also supported by the only known structure of a member of the PsrC/NrfD family (Jormakka et al., 2008). However, it is presently unknown whether methyl-menaquinone6 plays a role in sulphite respiration. In the context of the low midpoint potential of the redox pair sulphite/sulphide (– 116 mV) it is reasonable that a low-potential menaquinone derivative like methyl-menaquinol might be the electron donor to MccD. However, the capacity of the FdhABC complex to reduce methyl-menaquinol6 has not been investigated. In Fig. 4, a proton per electron ratio of 1 is assumed for the electron transport from formate to sulphite via the respiratory Fdh and Mcc systems. According to this model, formate oxidation coupled to quinone reduction is electrogenic using the redox loop mechanism while quinol oxidation by sulphite is not (Simon et al., 2008). Given the small amount of available free energy, a proton per electron ratio of less than 1 cannot be excluded, similar to what was experimentally suggested for polysulphide respiration (Klimmek et al., 2004).
Function of MccA and organization of mcc gene clusters in other bacteria
Apart from W. succinogenes, the Gammaproteobacterium S. oneidensis MR-1 is the best-studied bacterium with respect to MccA function. The transcription of the S. oneidensis mcc gene cluster was shown to be upregulated in the presence of thiosulphate (Beliaev et al., 2005). Because this organism is able to reduce thiosulphate to yield sulphite, it is conceivable that the observed regulatory effect might have been mediated by sulphite (Burns and DiChristina, 2009). In line with this hypothesis, S. oneidensis MccA (recently renamed SirA) was shown to be involved in respiratory sulphite reduction and SirA was identified as the sulphite reductase on the basis of activity staining in a gel by Shirodkar et al. (2011). These authors also reported that the presence/absence of mcc (sir) gene clusters in different Shewanella species correlated with their ability/disability to reduce sulphite. To our knowledge, W. succinogenes and S. oneidensis are the only organisms for which physiological and/or biochemical data on MccA function are available to date.
The 42 known mccA (sirA) genes are encoded in one or two copies as part of mcc gene clusters in genomes of the epsilonproteobacterial genera Wolinella (1 single mccA gene copy), Sulfurospirillum (1 single copy) and Campylobacter (11 single copies); the gammaproteobacterial genera Edwardsiella (3 single copies), Ferrimonas (2 gene copies in one genome) and Shewanella (19 genes; 11 single copies; 4 double copies); the deltaproteobacterial genus Anaeromxyobacter (3 single copies); and in two betaproteobacterial genomes in the order Burkholderiales (single copies) (Fig. S4). Each locus contains a MccB-type peptidyl-prolyl cis-trans isomerase and a cytochrome c haem synthase (either of the CcsA1-type or the NrfEFG-type depending on the presence of the cytochrome c biogenesis system I or II) (Hartshorne et al., 2007; Kranz et al., 2009). A S. oneidensis mutant that lacked the nrfG homologue (sirG) was unable to reduce sulphite suggesting that the corresponding protein is involved in MccA maturation similar to W. succinogenes CcsA1 (Shirodkar et al., 2011). Of the mcc loci, 26 encode an MccCD quinol dehydrogenase module. Constitutive expression of the mccCD (sirCD) genes in S. oneidensis was recently shown to compensate the lack of CymA, which acts as a versatile membrane-bound tetrahaem cytochrome c quinol dehydrogenase that directly or indirectly delivers electrons to various terminal reductases of anaerobic respiration (Cordova et al., 2011). This result indicated that MccCD (SirCD) is functional and supports the finding that S. oneidensis sulphite respiration is independent of CymA (Shirodkar et al., 2011). Bacteria lacking the mccCD/sirCD genes might functionally compensate this deficit by using alternative proteins encoded elsewhere on their genomes. In principal, the Mcc/Sir systems of W. succinogenes and S. oneidensis are assumed to have equivalent functions. However, the precise role of the MccC and MccD homologues and the nature of the quinone species involved in electron transport of MccA needs to be elucidated for both bacteria in the future. Apart from the genes discussed above, there are several more genes that are present only occasionally in mcc gene clusters (Fig. S4). The function of these genes is not known and corresponding mutant strains have not been described.
Evolutionary history of MccA
Phylogenetic analysis of MccA protein sequences in context with sequences of other multihaem cytochromes c revealed a surprisingly close relationship with a rather diverse family of octa- and tetrahaem cytochromes c encoded in the genomes of predominantly Deltaproteobacteria as well as species of Veruccomicrobia, Planctomycetes and Beta- and Gammaproteobacteria (Fig. 5A and B). The tetrahaem cytochromes c of the latter three are known as cytochrome c554 proteins. Studied cytochrome c554 proteins function as catabolic redox shuttles in nitrification but have also been shown to possess nitric oxide reductase activity associated with the haem c group bound to the second haem c binding motif (Upadhyay et al., 2006; Klotz and Stein, 2011). An overwhelming number of these cytochrome c554 proteins, encoded in deltaproteobacterial genomes, have not been characterized. Interestingly, cytochrome c554-encoding genes appear to be absent from epsilonproteobacterial genomes.
Figure 5. Phylogenetic relationships within the superfamily of multihaem cytochromes c. A. Consensus tree constructed after Bayesian inference of phylogeny from the ClustalX alignments of 285 NrfA, Onr, Hao/Hzo, cytochrome c554, MccA and Otr protein sequences. The N- and C-terminal sequences outside the trihaem core haem c binding motifs were eliminated from the final alignment that was subjected to Bayesian inference of phylogeny using the BEAST package (see Experimental procedures). An unrooted 50% majority rule consensus phylogram was displayed as a circle tree, for which posterior probability values of the main nodes are shown. Mean branch lengths are characterized by a scale bar indicating the evolutionary distance between the proteins (changes per amino acid position). The branches are annotated with labels indicating the source organism and the protein sequence accession number. Clusters of sequences were categorized as tetrahaem (TCC; cytochrome c554 in teal), pentahaem (PCC; NrfA in green) or octahaem cytochromes c (OCC; Onr in brown, Hao/Hzo in red/gold, OTR in black, cytochrome c554 in teal and MccA in magenta) with clades of sequences from Epsilonproteobacteria shown in blue. B. Star tree configuration of (A) indicating that the clades coalesce in a node that separates a superclade of MccA/cytochrome c554 and Otr sequences from the Hao/Hzo, Onr and NrfA clades. C. Alignment of haem c binding motifs in multihaem cytochrome c families indicating the trihaem core (see text for details).
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Other multihaem cytochromes c that are reactive with sulphur and/or nitrogen compounds comprise the octahaem tetrathionate reductase (Otr), members of the octahaem hydroxylamine oxidoreductase/hydrazine oxidoreductase (Hao/Hzo) family as well as pentahaem cytochrome c (PCC) nitrite reductase (NrfA, see Introduction) and the related octahaem cytochrome c nitrite reductase (Onr) (Simon et al., 2011). A scenario for the evolution of this superfamily of multihaem cytochromes was reported recently by Klotz et al. (2008) based on NrfA, Onr and Hao/Hzo sequences and a putative evolutionary relationship between Hao and cytochrome c554 proteins had been suggested (but not tested) based on protein structure and haem c arrangements (Hooper et al., 2005). Inclusion of an extensive number of MccA, cytochrome c554 and Otr protein sequences in an updated phylogenetic analysis of the NrfA, Onr and Hao/Hzo proteins resulted in a phylogenetic tree that separated three major clades (the MccA/cytochrome c554 proteins, the Otr proteins and the Hao/Hzo/Onr/NrfA proteins), which are connected at the level of octahaem cytochrome c (OCC) proteins (Fig. 5B). All known Hao/Hzo, MccA and Otr proteins are OCC proteins. Interestingly, the detailed tree (Fig. 5A) reveals that the history of the cytochrome c554 family included three independent events of gene/protein truncation from OCC to tetrahaem cytochrome c (TCC) thereby creating several subfamilies whose emergence was likely tied to functional pressures. While the function of the three deltaproteobacterial tetrahaem cytochrome c554 protein subfamilies is experimentally untested, the tetrahaem cytochrome c554 protein subfamily represented in aerobic ammonia-oxidizing bacteria is known to be part of catabolic electron flow. In contrast, one of the two cytochrome c554 proteins encoded in anaerobic ammonia-oxidizing bacteria (CAJ75043), the genomic background in which nitrogen-based catabolic electron flow probably emerged (Klotz and Stein, 2011 and references therein), is a OCC protein. This apparent scenario of reduction in participating haems within the MccA/cytochrome c554 branch of the superfamily led us to propose a slightly revised scenario for the evolutionary relationships. While not contradicting previous findings that led to a proposal of an evolutionary direction from PCC (NrfA) to OCC (Onr, Hao), the extended dataset suggests that the great diversity of extant proteins originated from an ancestral OCC protein that very likely was able to reduce a sulphur compound like sulphite or tetrathionate (as in extant MccA and Otr proteins). Divergence over evolutionary time has then led to new characteristics whereby modified and de novo-evolved active sites successfully reduced nitrogen oxides and/or oxyanions (cytochrome c554; Hao/Hzo, Onr, NrfA and Otr). Across the entire superfamily, it appears that only a subset of the Hao/Hzo family acquired the ability to oxidize hydroxylamine or hydrazine, utilizing disproportionation chemistry enabled by a covalently bonded circular symmetric trimer configuration (Hooper et al., 2005; Kostera et al., 2008). None of the enzymes in the Hao/Hzo family is known to oxidize sulphur compounds. The functional driver in the evolution of the MccA protein family might have been the ability to provide additional respiratory capacity paired with enriching the pool of reduced sulphur. Apparently, this development occurred exclusively in Proteobacteria and prevented emergence of the capacity to reduce nitrite. As illustrated in Fig. 5C, all members of the multihaem cytochrome c superfamily share a core of three haem c binding motifs including the one that binds the active site haem group in NrfA (haem 1), Onr (haem 4), Hao/Hzo (haem 4) and Otr (haem 2). This motif aligns with haem 2 of MccA and cytochrome c554. It is currently unclear which haem c group of MccA might be situated close to the active of sulphite reduction or which role the CX15CH-ligated haem c group of MccA (haem 8) might play apart from being essential for protein integrity.