Critical Review Cytoplasmic Sulfur Trafficking in Sulfur-Oxidizing Prokaryotes
Christiane Dahl*
€ r Mikrobiologie & Biotechnologie, Rheinische Friedrich-WilhelmsInstitut fu Universita€t Bonn, Bonn, Germany
Abstract Persulfide groups are chemically versatile and participate in a wide array of biochemical pathways. Although it is well documented that persulfurated proteins supply a number of important and elaborate biosynthetic pathways with sulfane sulfur, it is far less acknowledged that the enzymatic generation of persulfidic sulfur, the successive transfer of sulfur as a persulfide between multiple proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential steps during dissimilatory sulfur oxidation in bacteria and archaea. Here,
the currently available information on sulfur trafficking in sulfur oxidizing prokaryotes is reviewed, and the idea is discussed that sulfur is always presented to cytoplasmic oxidizing enzymes in a protein-bound form, thus preventing the occurrence of free sulfide inside of the prokaryotic cell. Thus, sulfur trafficking emerges as a central element in sulfuroxidizing pathways, and TusA homologous proteins appear to C 2015 be central and common elements in these processes. V IUBMB Life, 67(4):268–274, 2015
Keywords: dissimilatory sulfur oxidation; Dsr proteins; sulfur-oxidizing prokaryotes; sulfur trafficking; heterodisulfide reductase-like system; TusA; DsrE; DsrC; thiosulfate transfer; Allochromatium vinosum; Metallosphaera cuprina
Introduction Sulfur transfer reactions play an important role in a number of biosynthetic pathways such as the thiolation of tRNA and the synthesis of iron–sulfur clusters, thiamine, molybdopterin cofactor, biotin, or lipoic acid (1,2). It is well established that sulfur delivery to these pathways involves the successive transfer of sulfur as a persulfide between multiple proteins, many of which are highly conserved across species. In contrast, it is far less acknowledged that the enzymatic generation of persul-
Abbreviations: Hdr, heterodisulfide reductase; Dsr, dissimilatory sulfite reductase; GSSH, glutathione persulfide C 2015 International Union of Biochemistry and Molecular Biology V Volume 67, Number 4, April 2015, Pages 268–274 € r Mikrobiologie & *Address correspondence to: Christiane Dahl, Institut fu Biotechnologie, Rheinische Friedrich-Wilhelms-Universita€t Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany. Tel.: 149–228-732119. Fax: 149–228-737576. E-mail: [email protected] Received 19 December 2014; Accepted 27 February 2015 DOI 10.1002/iub.1371 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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fidic sulfur, the transfer of sulfane sulfur as persulfide between different proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential components of sulfur oxidation pathways that are linked to energy transformation via photosynthesis or respiratory processes (3–5). In these dissimilatory pathways, reduced sulfur compounds serve as electron donors for anaerobic phototrophic and aerobic or anaerobic chemotrophic growth and are mostly oxidized to sulfate (6,7). Sulfur-oxidizing prokaryotes are very diverse, and a universal mechanism for the oxidation of reduced sulfur compounds does not exist (6,7). Complete oxidation pathways such as the Sox pathway of thiosulfate oxidation typically found in facultative lithotrophs like the Alphaproteobacterium Paracoccus pantotrophus may run in the periplasm (8,9). On the other hand periplasmic as well as cytoplasmic steps are involved in a vast number of prokaroytes specialized on lithotrophic growth on reduced sulfur compounds (6,7,10,11). Information about sulfur transfer steps immediately involved in sulfur oxidation reactions taking place outside of the cytoplasm, for example, in the periplasmic space of Gram-negative bacteria, is scarce, and this review will therefore focus on sulfur trafficking relevant for those steps of dissimilatory sulfur oxidation occurring in the bacterial cytoplasm.
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FIG 1
A: Model for sulfur oxidation in Allochromatium vinosum via the Dsr pathway integrating a sulfur-mobilizing function for Rhd, sulfur transfer functions for TusA and DsrEFH, and a substrate-donating function for DsrC. As detailed in the text, the model is based on biochemical as well as on molecular genetic evidence. B: Model for sulfur oxidation in Metallosphaera cuprina via the pathway including the Hdr-like complex. The model integrates thiosulfate-mobilizing and thiosulfate-accepting functions for DsrE3A and TusA, respectively. The formation of protein-S-thiosulfonates and thiosulfate transfer as indicated is based on experimental evidence, whereas the sulfite-generating function of the Hdr-like complex is derived from database analyses and transcriptomic profiling studies. Biochemical or molecular genetic data are completely lacking at present. Additional reactions are possible for DsrE3A and TusA (11) that are not depicted here for clarity.
Sulfur Oxidation in the Periplasm The periplasmic Sox pathway is a prime example for oxidation of protein-bound sulfur atoms (8,9). The heterodimeric SoxYZ protein acts as the central player and carries pathway intermediates covalently bound to a cysteine residue located near the carboxy-terminus of the SoxY subunit (12–14). The c-type cytochrome SoxXA catalyzes the oxidative formation of a disulfide linkage between the sulfane sulfur of thiosulfate and the cysteine of SoxY (15). The sulfone group is then hydrolytically released as sulfate in a reaction catalyzed by SoxB (16). In part of the organisms harboring Sox proteins, the next step is oxidation of the SoxY-bound sulfane sulfur to a sulfone by the hemomolybdoprotein SoxCD and again hydrolytic release of sulfate (9). In another group of sulfur oxidizers, SoxCD is not present, and it has been suggested that SoxL, a protein con-
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taining a typical rhodanese (i.e., thiosulfate:acceptor sulfur transferase) domain, catalyzes the transfer of SoxY-bound sulfane sulfur to zero-valent sulfur that is stored as an intermediate either in extracellular or in intracellular sulfur globules (17). However, conclusive biochemical or genetic evidence for this proposal has not yet been provided.
Sulfur Oxidation in the Cytoplasm Currently, the best studied of the sulfur oxidation pathways operating in the cytoplasm is the so-called Dsr pathway (Fig. 1A) involving the enzyme reverse dissimilatory sulfite reductase (DsrAB; refs. 18 and 19). It is pursued by many environmentally important photolithoautotrophic and chemolithoautotrophic bacteria and typically includes the accumulation of
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zero-valent sulfur as a transient product during the oxidation of sulfide, thiosulfate, or polysulfides (6,7). Low-molecularweight organic persulfides such as glutathione amide persulfide have been proposed as carrier molecules transferring sulfur from the periplasmic or extracellular sulfur deposits into the cytoplasm (7). It is not yet known how exactly the proposed persulfidic carrier molecules are generated and whether specific enzymes are involved in this process nor have transporters for such molecules be characterized from any sulfuroxidizing prokaryote. An extensive sulfur-trafficking network, including a rhodanese and the proteins TusA, DsrE2, and DsrEFH, then delivers the sulfane sulfur to the final sulfuraccepting protein DsrC, which in its persulfurated form most likely serves as a direct substrate for DsrAB (3). This enzyme catalyzes the formation of sulfite. In a wide array of chemotrophic and also phototrophic sulfur oxidizers that do not contain the Dsr pathway, the gene cluster hdrC1B1A1orf2hdrC2B2 is present that encodes a possible heterodisulfide reductase (Hdr)-like complex. Hdrs are enzymes present in methanogenic archaea and catalyze the reduction of the heterodisulfide, CoM-S-S-CoB, formed in the last step of methanogenesis (20,21). The Hdr-like complex in sulfur oxidizers is predicted to have a completely different function and to be involved in a process functionally replacing the Dsr system (Fig. 1B; refs. 22 and 23). The hdr-like genes are inevitably linked to genes encoding TusA, a DsrE family protein and often also rhodanese homologs. Recently, biochemical evidence was provided for transfer of thiosulfate mediated by DsrE/TusA homologs from the thermoacidophilic archaeon Metallosphaera cuprina (11). Genes for liponamidebinding proteins resembling protein H of the glycine cleavage system and several proteins responsible for the biosynthesis of liponamide-binding proteins are also always part of or reside in immediate vicinity of the tusA-dsrE-hdr clusters pointing at the possibility that a liponamide-binding protein takes active part in the reaction catalyzed by the Hdr-like proteins (11). From the above, it is concluded that TusA, DsrE homologs, and in many cases also a cytoplasmic rhodanese are common elements in bacterial as well as in archaeal dissimilatory sulfur oxidizers. In the following, the current knowledge on the role of these sulfur transferases during sulfur oxidation in prokaryotes is reviewed, and the suggestion is promoted that sulfane sulfur is generally delivered to cytoplasmic sulfitegenerating systems in protein-bound form. We furthermore discuss the possibility that the protein TusA is an essential and central player in these processes.
The TusA Protein TusA (cd00291) is a highly conserved, widely distributed 8kDa protein that may be found as a single protein or fused to other proteins such as cysteine desulfurases or rhodaneses (4,24,25). TusA has been extensively studied both structurally and functionally (25,26). All TusA proteins contain a common Cys-Pro-X-Pro sequence motif in the N-terminal region with
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the sulfane sulfur-binding cysteine as the central element (4,11,25). In E. coli, the motif contains a glutamate residue at position “X,” which is replaced by a hydrophobic residue (most often leucine, sometimes isoleucine or methionine) or glycine in the proteins from sulfur oxidizers (4). Evidence is emerging that the nonconserved amino acid in the motif is an element decisive for interaction of TusA with its various possible protein partners (4,24). TusA alone is incapable of mobilizing sulfur from thiosulfate or low-molecular-weight organic persulfides like glutathione persulfide (GSSH; ref. 4). For E. coli, it is well established that TusA functions as a sulfur mediator for the synthesis of 2-thiouridine of the modified wobble base 5-methyl-aminomethyl-2-thiouridine [(mnm)5s2U] in tRNA (27). Over the last years, several additional roles have been assigned to TusA. A tusA-deficient E. coli strain was found to form filamentous cells due to a severe impairment of FtsZ ring formation (28,29). Furthermore, TusA in conjunction with TusBCD and TusE appears to play a role in the maintenance of the intracellular redox state (30), and it has been suggested that TusA is involved in sulfur transfer for the synthesis of molybdopterin and also in the balanced regulation of the availability of IscS to various biomolecules in E. coli (24). Taken together, all these findings indicate a pleiotropic role for TusA in E. coli that involves specific interaction with several different protein partners (24). Bioinformatic analyses and transcriptomic profiling provided first hints that the role of TusA in bacteria other than E. coli is not limited to biosynthetic processes. In several sulfur oxidizers including Acidithiobacillus ferrooxidans, Metallosphaera sedula, and the purple sulfur bacterium Allochromatium vinosum, relative mRNA levels for tusA were significantly higher under sulfur-oxidizing conditions than in the absence of reduced sulfur compounds (22,31–33). An A. vinosum strain deficient of the rhd-tusA-dsrE2 genes was impaired in its ability to degrade zero-valent sulfur formed during the oxidation of sulfide and thiosulfate (4). The eminently important role of TusA is further highlighted by the finding that TusA is among the most abundant proteins in A. vinosum cells grown photolithoautotrophically on reduced sulfur compounds (Fig. 2; ref. 34). In these cells, enzymes involved in carbon dioxide fixation (RubisCo, phosphoribulokinase), photosynthesis (light-harvesting complexes), and energy conservation (subunits of ATP synthase) are very abundant. Furthermore, enzymes of oxidative sulfur metabolism like flavocytochrome c sulfide dehydrogenase (FccAB), DsrA, and DsrC and components of the Sox system dominate among the highest ranking proteins. It is intriguing that even SoxYZ and DsrC, the sulfur substrate donor proteins in the Sox and Dsr systems, respectively, are outnumbered by TusA (Fig. 2).
The Input Modules In many sulfur oxidizers, the tusA gene is flanked in the same direction of transcription by two genes encoding a rhodaneselike protein (rhd) and a protein of the DsrE superfamily
Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
FIG 2
The 150 most abundant proteins in A. vinosum grown with sulfur as electron donor. Proteins are ranked according to the ratio of their peptide spectrum matches and the number of amino acids thereby avoiding an underestimation of small and overestimation of large proteins. Proteins belonging to five functional groups were distinguished: energy conservation (blue); C-metabolism (green); gene expression, replication, and chaperones (gray); photosynthesis (red); and sulfur metabolism (yellow). Data taken from ref. 34.
(dsrE2; ref. 23). In fact, the rhd-tusA-dsrE2 or at least a tusAdsrE2 arrangement occurs not only in all currently genome sequenced phototrophic sulfur oxidizers harboring the Dsr system but also in a wide array of chemotrophic and also phototrophic sulfur bacteria containing the hdrC1B1A1orf2hdrC2B2 gene cluster. The latter include not only bacteria belonging to the Aquificaceae or the genera Acidithiobacillus and Thioalkalivibrio but also archaeal sulfur oxidizers of the genera Metallosphaera, Acidianus, and Sulfolobus. Inevitably, in these organisms, the tusA-dsrE2 genes are immediately linked with the genes encoding the Hdr-like protein complex. Genetic linkage of the rhd-tusA-dsrE2 set with dsr genes is also seen, for example, in representatives of the green sulfur bacterial family Chlorobiaceae (23). The rhd-tusA-dsrE2 genes are cotranscribed in A. vinosum (4). In this as well as in all other cases studied, the genes follow the same pattern of transcription as observed for the established or putative cytoplasmic sulfane sulfur-oxidizing proteins (i.e., the Dsr or Hdr-like system) and other proteins involved in oxidative sulfur metabolism such as sulfur globule proteins or the enzymes of the two different cytoplasmic sulfite oxidation pathways in A. vinosum (4,22,32,33,35). Taken together, the above considerations indicate rhd and possibly also dsrE2-encoded proteins as entry points for sulfur delivery to TusA. Indeed, biochemical evidence supporting this notion was recently provided for two different and independent systems (Figs. 1 and 3; refs. 4 and 11). The rhd-encoded 12-kDa protein consists of a single rhodanese (thiosulfate:cyanide sulfur transferase) domain (pfam00581) and bears resemblance to the cytoplasmic rhodanese GlpE from E. coli (36). Single-catalytic rhodanese domain
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proteins are encoded by both eukaryotic and prokaryotic genomes and exert various physiological functions (37). The protein from A. vinosum (Alvin_2599) is a soluble monomer and catalyzes sulfur transfer from thiosulfate or GSSH to cyanide in vitro. Mass spectrometry verified the transfer of sulfane sulfur from both substrates to the conserved active site of cysteine but not to a second only partly conserved cysteine (4). Most importantly, TusA was clearly established as a protein accepting sulfane sulfur from the A. vinosum rhodanese. It is therefore conceivable to assume that this protein also mediates sulfur transfer from low-molecular-weight organic perthiols to TusA in vivo (Fig. 1A). Current suggestions for oxidative sulfur metabolism in the genus Acidithiobacillus include the occurrence of persulfide sulfur and/or thiosulfate as intermediates in the cytoplasm (10,35). As thiosulfate also serves as a substrate for Rhd_2599 in vitro, sulfur input into TusA from thiosulfate via the rhodanese cannot be completely excluded for the in vivo situation (Fig. 3). The latter sulfur input pathway into TusA is, however, highly unlikely in sulfur oxidizers that harbor the periplasmic Sox system which would be expected to effectively oxidize all externally available thiosulfate before entry into the cytoplasm. All DsrE family proteins (COG1553 within the DsrE superfamily cl00672 and pfam13686) have one cysteine in common that corresponds to the active site cysteine of the DsrE and TusD subunits of the structurally characterized DsrEFH and TusBCD proteins from A. vinosum and E. coli, respectively (5,38). Many sulfur oxidizers contain more than one copy of dsrE-like genes. The archaeon M. cuprina is a prominent example. Three of its four dsrE copies reside next to genes encoding TusA homologs (11). This finding prompted the attempt to sort DsrE-like proteins into groups based on sequence similarity and cysteine residues conserved within the groups (11). The A. vinosum protein (Alvin_2601)-encoded downstream of the rhd-tusA genes falls into group DsrE2A with the active site cysteine present as the second in a conserved Cys-X9-Cys motif. Recombinant A. vinosum DsrE2A is an experimentally established membrane protein with two
FIG 3
TusA as the central element of cytoplasmic sulfur trafficking in sulfur-oxidizing prokaryotes. Established pathways are indicated by black arrows. Gray arrows highlight proposed pathways for which biochemical and molecular genetic evidence is not available at present.
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predicted transmembrane helices arranged such that the carboxy-terminal part of the protein carrying one strictly conserved and two further cysteine residues is located in the cytoplasm (4). The same topology is predicted for the DsrE2A proteins encoded in all members of the Chromatiaceae and Acidithiobacillaceae families listed in ref. 23. Sulfur transferase activity could so far not be proven for this protein. GSSH and thiosulfate cannot serve as sulfur donors for A. vinosum DsrE2A. It is persulfurated by incubation with sulfide in vitro; however, it has not yet been experimentally established whether the active site cysteine is the only residue capable of sulfur binding (4). Just as the DsrE2A protein from A. vinosum, the archaeal protein Mcup_0682 from M. cuprina does not react with GSSH and thiosulfate in vitro (11). The archaeal protein belongs to a different subgroup (DsrE2B) and is an established soluble protein. In contrast to A. vinosum DsrE2A, this protein is not even persulfurated in the presence of sulfide. In summary, the role of DsrE2 proteins remains elusive at present. However, the analysis of M. cuprina DsrE3A (Mcup_681), a protein with a conserved Cys-X7-Cys motif with the active site cysteine as the first residue, provided major insights and suggested a completely new pathway of sulfur input into TusA (Figs. 1 and 3; ref. 11). Both, the archaeal DsrE3A and the TusA protein proved to react with tetrathionate but not with sulfide, GSSH, polysulfide, or thiosulfate. The products were identified as protein-Cys-S-thiosulfonates. MALDI-TOF mass spectrometry confirmed thiosulfate transfer from DsrE3A-thiosulfonate to TusA (11). The back reaction, that is, the transfer of thiosulfate from thiosulfonated TusA to DsrE3A, was not possible. The reaction of tetrathionate with DsrE3A is feasible in vivo. M. cuprina is known to grow on tetrathionate but not on thiosulfate (39) albeit transporters mediating import of tetrathionate into the cytoplasm have not been identified so far (40,41). Altogether, these observations imply that DsrE3A functions as a thiosulfate donor to TusA in vivo. Thiosulfonated TusA could then serve as the substrate for other enzymes such as the Hdr-like system (see below).
The Output Modules Here, the idea is put forward that two principally different output modules exist in sulfur-oxidizing prokaryotes that achieve the oxidation of TusA-bound sulfane sulfur (Fig. 3). The first is the experimentally well-documented delivery of sulfur to reverse-acting dissimilatory sulfite reductase (DsrAB) and the second is the proposed oxidation by an Hdr-like system. The analysis of the sulfur oxidation pathway in A. vinosum revealed major similarities between the E. coli Tus sulfur relay system for tRNA modification and several of the Dsr proteins (5,42). DsrH, DsrF, and DsrE share sequence similarity with TusB, TusC, and TusD, respectively (27). Furthermore, DsrC exhibits sequence similarities with the E. coli protein TusE (27,42). In A. vinosum, DsrEFH and DsrC are encoded in the same gene cluster as DsrAB (18,19). In E. coli, TusA, TusBCD, and TusE act as sulfur transferases and mediate sulfur transfer between the cysteine desulfurase IscS and MnmA, a dedicated
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thiouridylase (27). In a highly organized series of events, the conserved cysteine in the amino-terminus of TusA (Cys19 in the E. coli protein) is first persulfurated by IscS. The persulfide is then transferred to the TusD subunit of TusBCD. Most probably sulfur transfer from TusD to TusE is the next step. Using the persulfide sulfur of TusE, MnmA finally catalyzes ATP-dependent 2thiouridine formation at position 34 of the tRNA (27). Like TusBCD from E. coli, DsrEFH from A. vinosum is a hexameric protein arranged in a a2b2c2 structure (5). It harbors two conserved cysteine residues in the active sites of the DsrE (DsrE-Cys78) and the DsrH (DsrH-Cys20) subunits. Although both of these cysteine residues are essential for sulfur oxidation in vivo, sulfur is only bound to DsrE-Cys78 in vitro. DsrE-Cys78 exactly corresponds to the active site of cysteine residue of E. coli TusD (5). DsrH-Cys20 is not persulfurated on incubation with sulfide (3). DsrEFH on its own is incapable of mobilizing sulfur from persulfidic molecules or thiosulfate (3) and therefore needs a donor protein. A. vinosum TusA is a firmly established interaction partner of DsrEFH. The presence of the conserved active site of cysteine residues in TusA and in the DsrE subunit proved to be essential for the interaction. Sulfur transfer between TusA and DsrEFH is reversible (4). The TusE homolog DsrC is an eminently important 12- to 14-kDa protein that works as the physiological partner of the DsrAB sulfite reductase not only in sulfur-oxidizing prokaryotes but also in sulfate-reducing prokaryotes (23). DsrC is a member of the DsrC/TusE/RpsA superfamily and contains two strictly conserved redox active cysteines in a flexible carboxyterminal arm (42): CysA is the penultimate residue at the Cterminus, and CysB is located about 10 residues upstream. TusE proteins contain only CysA (23). DsrEFH and DsrC from A. vinosum strongly interact in vitro. When combined in solution in their native, nonpersulfurated state, DsrEFH and DsrC form a tight complex as shown by native polyacrylamide gel electrophoresis (3). Each DsrE2F2H2 heterohexamer associates with either one or two DsrC molecules. Interaction of DsrEFH with DsrC is strictly dependent on the presence of DsrE-Cys78 and DsrC-CysA (3,42). Persulfurated DsrEFH transfers sulfur atoms specifically to CysA of A. vinosum DsrC but not to CysB, which was shown by the use of DsrC variants lacking either one or both cysteine residues (3). DsrH-Cys20 is not required for sulfur transfer from DsrEFH to DsrC. The reverse reaction, that is, sulfur transfer from DsrC to DsrEFH was not detected. Persulfurated DsrC serves as the substrate for DsrAB, and the oxidation of DsrC-CysA-S2 by this enzyme is thought to result in persulfonated DsrC (DsrC-CysA-S032) from which sulfite is possibly released by the formation of a disulfide bridge between CysA and CysB (Fig. 1A). The oxidation of sulfite, the product of the DsrAB-catalyzed reaction in sulfur oxidizers, to the final product sulfate is performed either indirectly by APS reductase and ATP sulfurylase via adenosine-50 -phosphosulfate (APS) or directly via the cytoplasmically oriented membrane-bound iron– sulfur molybdoenzyme SoeABC (43).
Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
Recently, a broad database survey revealed a mutually exclusive occurrence of genes encoding Dsr and Hdr-like complex proteins in genome-sequenced sulfur-oxidizing prokaryotes, thus strongly supporting earlier suggestions that the putative Hdr-like complex is involved in a process functionally replacing the Dsr system (22). The immediate linkage of rhd-tusA-dsrE2 or tusA-dsrE2 gene arrangements with the strictly conserved hdrC1B1A1orf2hdrC2B2 gene set in all prokaryotes containing the hdr-like genes in conjunction with the experimentally documented transfer of thiosulfate from M. cuprina DsrE3A to TusA further promotes the notion that an Hdr-like protein complex is involved in the generation of sulfite from TusA-bound sulfur compounds. In species of the genus Acidithiobacillus, organic persulfides that are formed as intermediates during the oxidation of externally available elemental sulfur to sulfite have been suggested as substrates for the sulfur-oxidizing enzymes (44). The Hdr-like system could serve as such a system (10,22,35) and is proposed here to also include TusA as a central element. In archaea like M. cuprina, thiosulfonated TusA could serve as the substrate for the Hdr-like complex (11). In this case, it can be envisioned that the sulfonate group is first released, either hydrolytically as sulfate or reductively as sulfite, by an as yet unknown mechanism. The sulfane group remaining on TusA could then be oxidized and finally also be released (11).
Conclusions In summary, it is evident that protein-mediated sulfur transfer and its delivery to the sulfite-generating enzymes in proteinbound form are of pivotal importance for dissimilatory sulfur oxidation. Extended analyses of arrangements of genes involved in sulfur oxidation confirm not only a tight linkage of (rhd-)tusAdsrE2 with genes encoding major components of the sulfur oxidation machinery in many cases but also indicate a near ubiquitous occurrence of these genes in sulfur oxidizers. The protein TusA appears to be a common element in sulfur oxidizers, and a central role of this protein is proposed. A rhodanese-like protein is needed to mobilize sulfur from lowmolecular-weight organic perthiols and transfers it via TusA (and possibly also via DsrE2), either via DsrEFH and DsrC to DsrAB or directly to the Hdr-like complex (Fig. 3). An alternative route of sulfur input to TusA is exemplified by the archaeal DsrE3A protein that reacts directly with tetrathionate and transfers thiosulfate onto TusA. Finally, DsrAB and alternatively the proposed Hdr-like complex function as the sulfite producing entities. The latter may work only in conjunction with further, so far unidentified, proteins.
Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (Grant Da 351/6-1).
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[22] Quatrini, R., Appia-Ayme, C., Denis, Y., Jedlicki, E., Holmes, D. S., et al. (2009) Extending the models for iron and sulfur oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. BMC Genomics 10, 394. doi: 10.1186/1471-2164-10-394. [23] Venceslau, S. S., Stockdreher, Y., Dahl, C., and Pereira, I. A. C. (2014) The “bacterial heterodisulfide” DsrC is a key protein in dissimilatory sulfur metabolism. Biochim. Biophys. Acta 1837, 1148–1164. [24] Dahl, J. U., Radon, C., Buhning, M., Nimtz, M., Leichert, L. I., et al. (2013) The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis. J. Biol. Chem. 288, 5426–5442. [25] Shi, R., Proteau, A., Villarroya, M., Moukadiri, I., Zhang, L. H., et al. (2010) Structural basis for Fe–S cluster assembly and tRNA thiolation mediated by IscS protein–protein interactions. PLoS Biol. 8, e1000354. doi: 10.1371/ journal.pbio.1000354. [26] Katoh, E., Hatta, T., Shindo, H., Ishii, Y., Yamada, H., et al. (2000) High precision NMR structure of YhhP, a novel Escherichia coli protein implicated in cell division. J. Mol. Biol. 304, 219–229. [27] Ikeuchi, Y., Shigi, N., Kato, J., Nishimura, A., and Suzuki, T. (2006) Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol. Cell 21, 97–108. [28] Yamashino, T., Isomura, M., Ueguchi, C., and Mizuno, T. (1998) The yhhP gene encoding a small ubiquitous protein is fundamental for normal cell growth of Escherichia coli. J. Bacteriol. 180, 2257–2261. [29] Ishii, Y., Yamada, H., Yamashino, T., Ohashi, K., Katoh, E., et al. (2000) Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli. Biosci. Biotechnol. Biochem. 64, 799–807. [30] Nakayashiki, T., Saito, N., Takeuchi, R., Kadokura, H., Nakahigashi, K., et al. (2013) The tRNA thiolation pathway modulates the intracellular redox state in Escherichia coli. J. Bacteriol. 195, 2039–2049. [31] Quatrini, R., Appia-Ayme, C., Denis, Y., Ratouchniak, J., Veloso, F., et al. (2006) Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling. Hydrometallurgy 83, 263–272. [32] Auernik, K. S., and Kelly, R. M. (2008) Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes. Appl. Environ. Microbiol. 74, 7723–7732. [33] Weissgerber, T., Dobler, N., Polen, T., Latus, J., Stockdreher, Y., et al. (2013) Genome-wide transcriptional profiling of the purple sulfur bacterium Allochro-
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Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
Christiane Dahl*
€ r Mikrobiologie & Biotechnologie, Rheinische Friedrich-WilhelmsInstitut fu Universita€t Bonn, Bonn, Germany
Abstract Persulfide groups are chemically versatile and participate in a wide array of biochemical pathways. Although it is well documented that persulfurated proteins supply a number of important and elaborate biosynthetic pathways with sulfane sulfur, it is far less acknowledged that the enzymatic generation of persulfidic sulfur, the successive transfer of sulfur as a persulfide between multiple proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential steps during dissimilatory sulfur oxidation in bacteria and archaea. Here,
the currently available information on sulfur trafficking in sulfur oxidizing prokaryotes is reviewed, and the idea is discussed that sulfur is always presented to cytoplasmic oxidizing enzymes in a protein-bound form, thus preventing the occurrence of free sulfide inside of the prokaryotic cell. Thus, sulfur trafficking emerges as a central element in sulfuroxidizing pathways, and TusA homologous proteins appear to C 2015 be central and common elements in these processes. V IUBMB Life, 67(4):268–274, 2015
Keywords: dissimilatory sulfur oxidation; Dsr proteins; sulfur-oxidizing prokaryotes; sulfur trafficking; heterodisulfide reductase-like system; TusA; DsrE; DsrC; thiosulfate transfer; Allochromatium vinosum; Metallosphaera cuprina
Introduction Sulfur transfer reactions play an important role in a number of biosynthetic pathways such as the thiolation of tRNA and the synthesis of iron–sulfur clusters, thiamine, molybdopterin cofactor, biotin, or lipoic acid (1,2). It is well established that sulfur delivery to these pathways involves the successive transfer of sulfur as a persulfide between multiple proteins, many of which are highly conserved across species. In contrast, it is far less acknowledged that the enzymatic generation of persul-
Abbreviations: Hdr, heterodisulfide reductase; Dsr, dissimilatory sulfite reductase; GSSH, glutathione persulfide C 2015 International Union of Biochemistry and Molecular Biology V Volume 67, Number 4, April 2015, Pages 268–274 € r Mikrobiologie & *Address correspondence to: Christiane Dahl, Institut fu Biotechnologie, Rheinische Friedrich-Wilhelms-Universita€t Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany. Tel.: 149–228-732119. Fax: 149–228-737576. E-mail: [email protected] Received 19 December 2014; Accepted 27 February 2015 DOI 10.1002/iub.1371 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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fidic sulfur, the transfer of sulfane sulfur as persulfide between different proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential components of sulfur oxidation pathways that are linked to energy transformation via photosynthesis or respiratory processes (3–5). In these dissimilatory pathways, reduced sulfur compounds serve as electron donors for anaerobic phototrophic and aerobic or anaerobic chemotrophic growth and are mostly oxidized to sulfate (6,7). Sulfur-oxidizing prokaryotes are very diverse, and a universal mechanism for the oxidation of reduced sulfur compounds does not exist (6,7). Complete oxidation pathways such as the Sox pathway of thiosulfate oxidation typically found in facultative lithotrophs like the Alphaproteobacterium Paracoccus pantotrophus may run in the periplasm (8,9). On the other hand periplasmic as well as cytoplasmic steps are involved in a vast number of prokaroytes specialized on lithotrophic growth on reduced sulfur compounds (6,7,10,11). Information about sulfur transfer steps immediately involved in sulfur oxidation reactions taking place outside of the cytoplasm, for example, in the periplasmic space of Gram-negative bacteria, is scarce, and this review will therefore focus on sulfur trafficking relevant for those steps of dissimilatory sulfur oxidation occurring in the bacterial cytoplasm.
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FIG 1
A: Model for sulfur oxidation in Allochromatium vinosum via the Dsr pathway integrating a sulfur-mobilizing function for Rhd, sulfur transfer functions for TusA and DsrEFH, and a substrate-donating function for DsrC. As detailed in the text, the model is based on biochemical as well as on molecular genetic evidence. B: Model for sulfur oxidation in Metallosphaera cuprina via the pathway including the Hdr-like complex. The model integrates thiosulfate-mobilizing and thiosulfate-accepting functions for DsrE3A and TusA, respectively. The formation of protein-S-thiosulfonates and thiosulfate transfer as indicated is based on experimental evidence, whereas the sulfite-generating function of the Hdr-like complex is derived from database analyses and transcriptomic profiling studies. Biochemical or molecular genetic data are completely lacking at present. Additional reactions are possible for DsrE3A and TusA (11) that are not depicted here for clarity.
Sulfur Oxidation in the Periplasm The periplasmic Sox pathway is a prime example for oxidation of protein-bound sulfur atoms (8,9). The heterodimeric SoxYZ protein acts as the central player and carries pathway intermediates covalently bound to a cysteine residue located near the carboxy-terminus of the SoxY subunit (12–14). The c-type cytochrome SoxXA catalyzes the oxidative formation of a disulfide linkage between the sulfane sulfur of thiosulfate and the cysteine of SoxY (15). The sulfone group is then hydrolytically released as sulfate in a reaction catalyzed by SoxB (16). In part of the organisms harboring Sox proteins, the next step is oxidation of the SoxY-bound sulfane sulfur to a sulfone by the hemomolybdoprotein SoxCD and again hydrolytic release of sulfate (9). In another group of sulfur oxidizers, SoxCD is not present, and it has been suggested that SoxL, a protein con-
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taining a typical rhodanese (i.e., thiosulfate:acceptor sulfur transferase) domain, catalyzes the transfer of SoxY-bound sulfane sulfur to zero-valent sulfur that is stored as an intermediate either in extracellular or in intracellular sulfur globules (17). However, conclusive biochemical or genetic evidence for this proposal has not yet been provided.
Sulfur Oxidation in the Cytoplasm Currently, the best studied of the sulfur oxidation pathways operating in the cytoplasm is the so-called Dsr pathway (Fig. 1A) involving the enzyme reverse dissimilatory sulfite reductase (DsrAB; refs. 18 and 19). It is pursued by many environmentally important photolithoautotrophic and chemolithoautotrophic bacteria and typically includes the accumulation of
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zero-valent sulfur as a transient product during the oxidation of sulfide, thiosulfate, or polysulfides (6,7). Low-molecularweight organic persulfides such as glutathione amide persulfide have been proposed as carrier molecules transferring sulfur from the periplasmic or extracellular sulfur deposits into the cytoplasm (7). It is not yet known how exactly the proposed persulfidic carrier molecules are generated and whether specific enzymes are involved in this process nor have transporters for such molecules be characterized from any sulfuroxidizing prokaryote. An extensive sulfur-trafficking network, including a rhodanese and the proteins TusA, DsrE2, and DsrEFH, then delivers the sulfane sulfur to the final sulfuraccepting protein DsrC, which in its persulfurated form most likely serves as a direct substrate for DsrAB (3). This enzyme catalyzes the formation of sulfite. In a wide array of chemotrophic and also phototrophic sulfur oxidizers that do not contain the Dsr pathway, the gene cluster hdrC1B1A1orf2hdrC2B2 is present that encodes a possible heterodisulfide reductase (Hdr)-like complex. Hdrs are enzymes present in methanogenic archaea and catalyze the reduction of the heterodisulfide, CoM-S-S-CoB, formed in the last step of methanogenesis (20,21). The Hdr-like complex in sulfur oxidizers is predicted to have a completely different function and to be involved in a process functionally replacing the Dsr system (Fig. 1B; refs. 22 and 23). The hdr-like genes are inevitably linked to genes encoding TusA, a DsrE family protein and often also rhodanese homologs. Recently, biochemical evidence was provided for transfer of thiosulfate mediated by DsrE/TusA homologs from the thermoacidophilic archaeon Metallosphaera cuprina (11). Genes for liponamidebinding proteins resembling protein H of the glycine cleavage system and several proteins responsible for the biosynthesis of liponamide-binding proteins are also always part of or reside in immediate vicinity of the tusA-dsrE-hdr clusters pointing at the possibility that a liponamide-binding protein takes active part in the reaction catalyzed by the Hdr-like proteins (11). From the above, it is concluded that TusA, DsrE homologs, and in many cases also a cytoplasmic rhodanese are common elements in bacterial as well as in archaeal dissimilatory sulfur oxidizers. In the following, the current knowledge on the role of these sulfur transferases during sulfur oxidation in prokaryotes is reviewed, and the suggestion is promoted that sulfane sulfur is generally delivered to cytoplasmic sulfitegenerating systems in protein-bound form. We furthermore discuss the possibility that the protein TusA is an essential and central player in these processes.
The TusA Protein TusA (cd00291) is a highly conserved, widely distributed 8kDa protein that may be found as a single protein or fused to other proteins such as cysteine desulfurases or rhodaneses (4,24,25). TusA has been extensively studied both structurally and functionally (25,26). All TusA proteins contain a common Cys-Pro-X-Pro sequence motif in the N-terminal region with
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the sulfane sulfur-binding cysteine as the central element (4,11,25). In E. coli, the motif contains a glutamate residue at position “X,” which is replaced by a hydrophobic residue (most often leucine, sometimes isoleucine or methionine) or glycine in the proteins from sulfur oxidizers (4). Evidence is emerging that the nonconserved amino acid in the motif is an element decisive for interaction of TusA with its various possible protein partners (4,24). TusA alone is incapable of mobilizing sulfur from thiosulfate or low-molecular-weight organic persulfides like glutathione persulfide (GSSH; ref. 4). For E. coli, it is well established that TusA functions as a sulfur mediator for the synthesis of 2-thiouridine of the modified wobble base 5-methyl-aminomethyl-2-thiouridine [(mnm)5s2U] in tRNA (27). Over the last years, several additional roles have been assigned to TusA. A tusA-deficient E. coli strain was found to form filamentous cells due to a severe impairment of FtsZ ring formation (28,29). Furthermore, TusA in conjunction with TusBCD and TusE appears to play a role in the maintenance of the intracellular redox state (30), and it has been suggested that TusA is involved in sulfur transfer for the synthesis of molybdopterin and also in the balanced regulation of the availability of IscS to various biomolecules in E. coli (24). Taken together, all these findings indicate a pleiotropic role for TusA in E. coli that involves specific interaction with several different protein partners (24). Bioinformatic analyses and transcriptomic profiling provided first hints that the role of TusA in bacteria other than E. coli is not limited to biosynthetic processes. In several sulfur oxidizers including Acidithiobacillus ferrooxidans, Metallosphaera sedula, and the purple sulfur bacterium Allochromatium vinosum, relative mRNA levels for tusA were significantly higher under sulfur-oxidizing conditions than in the absence of reduced sulfur compounds (22,31–33). An A. vinosum strain deficient of the rhd-tusA-dsrE2 genes was impaired in its ability to degrade zero-valent sulfur formed during the oxidation of sulfide and thiosulfate (4). The eminently important role of TusA is further highlighted by the finding that TusA is among the most abundant proteins in A. vinosum cells grown photolithoautotrophically on reduced sulfur compounds (Fig. 2; ref. 34). In these cells, enzymes involved in carbon dioxide fixation (RubisCo, phosphoribulokinase), photosynthesis (light-harvesting complexes), and energy conservation (subunits of ATP synthase) are very abundant. Furthermore, enzymes of oxidative sulfur metabolism like flavocytochrome c sulfide dehydrogenase (FccAB), DsrA, and DsrC and components of the Sox system dominate among the highest ranking proteins. It is intriguing that even SoxYZ and DsrC, the sulfur substrate donor proteins in the Sox and Dsr systems, respectively, are outnumbered by TusA (Fig. 2).
The Input Modules In many sulfur oxidizers, the tusA gene is flanked in the same direction of transcription by two genes encoding a rhodaneselike protein (rhd) and a protein of the DsrE superfamily
Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
FIG 2
The 150 most abundant proteins in A. vinosum grown with sulfur as electron donor. Proteins are ranked according to the ratio of their peptide spectrum matches and the number of amino acids thereby avoiding an underestimation of small and overestimation of large proteins. Proteins belonging to five functional groups were distinguished: energy conservation (blue); C-metabolism (green); gene expression, replication, and chaperones (gray); photosynthesis (red); and sulfur metabolism (yellow). Data taken from ref. 34.
(dsrE2; ref. 23). In fact, the rhd-tusA-dsrE2 or at least a tusAdsrE2 arrangement occurs not only in all currently genome sequenced phototrophic sulfur oxidizers harboring the Dsr system but also in a wide array of chemotrophic and also phototrophic sulfur bacteria containing the hdrC1B1A1orf2hdrC2B2 gene cluster. The latter include not only bacteria belonging to the Aquificaceae or the genera Acidithiobacillus and Thioalkalivibrio but also archaeal sulfur oxidizers of the genera Metallosphaera, Acidianus, and Sulfolobus. Inevitably, in these organisms, the tusA-dsrE2 genes are immediately linked with the genes encoding the Hdr-like protein complex. Genetic linkage of the rhd-tusA-dsrE2 set with dsr genes is also seen, for example, in representatives of the green sulfur bacterial family Chlorobiaceae (23). The rhd-tusA-dsrE2 genes are cotranscribed in A. vinosum (4). In this as well as in all other cases studied, the genes follow the same pattern of transcription as observed for the established or putative cytoplasmic sulfane sulfur-oxidizing proteins (i.e., the Dsr or Hdr-like system) and other proteins involved in oxidative sulfur metabolism such as sulfur globule proteins or the enzymes of the two different cytoplasmic sulfite oxidation pathways in A. vinosum (4,22,32,33,35). Taken together, the above considerations indicate rhd and possibly also dsrE2-encoded proteins as entry points for sulfur delivery to TusA. Indeed, biochemical evidence supporting this notion was recently provided for two different and independent systems (Figs. 1 and 3; refs. 4 and 11). The rhd-encoded 12-kDa protein consists of a single rhodanese (thiosulfate:cyanide sulfur transferase) domain (pfam00581) and bears resemblance to the cytoplasmic rhodanese GlpE from E. coli (36). Single-catalytic rhodanese domain
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proteins are encoded by both eukaryotic and prokaryotic genomes and exert various physiological functions (37). The protein from A. vinosum (Alvin_2599) is a soluble monomer and catalyzes sulfur transfer from thiosulfate or GSSH to cyanide in vitro. Mass spectrometry verified the transfer of sulfane sulfur from both substrates to the conserved active site of cysteine but not to a second only partly conserved cysteine (4). Most importantly, TusA was clearly established as a protein accepting sulfane sulfur from the A. vinosum rhodanese. It is therefore conceivable to assume that this protein also mediates sulfur transfer from low-molecular-weight organic perthiols to TusA in vivo (Fig. 1A). Current suggestions for oxidative sulfur metabolism in the genus Acidithiobacillus include the occurrence of persulfide sulfur and/or thiosulfate as intermediates in the cytoplasm (10,35). As thiosulfate also serves as a substrate for Rhd_2599 in vitro, sulfur input into TusA from thiosulfate via the rhodanese cannot be completely excluded for the in vivo situation (Fig. 3). The latter sulfur input pathway into TusA is, however, highly unlikely in sulfur oxidizers that harbor the periplasmic Sox system which would be expected to effectively oxidize all externally available thiosulfate before entry into the cytoplasm. All DsrE family proteins (COG1553 within the DsrE superfamily cl00672 and pfam13686) have one cysteine in common that corresponds to the active site cysteine of the DsrE and TusD subunits of the structurally characterized DsrEFH and TusBCD proteins from A. vinosum and E. coli, respectively (5,38). Many sulfur oxidizers contain more than one copy of dsrE-like genes. The archaeon M. cuprina is a prominent example. Three of its four dsrE copies reside next to genes encoding TusA homologs (11). This finding prompted the attempt to sort DsrE-like proteins into groups based on sequence similarity and cysteine residues conserved within the groups (11). The A. vinosum protein (Alvin_2601)-encoded downstream of the rhd-tusA genes falls into group DsrE2A with the active site cysteine present as the second in a conserved Cys-X9-Cys motif. Recombinant A. vinosum DsrE2A is an experimentally established membrane protein with two
FIG 3
TusA as the central element of cytoplasmic sulfur trafficking in sulfur-oxidizing prokaryotes. Established pathways are indicated by black arrows. Gray arrows highlight proposed pathways for which biochemical and molecular genetic evidence is not available at present.
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predicted transmembrane helices arranged such that the carboxy-terminal part of the protein carrying one strictly conserved and two further cysteine residues is located in the cytoplasm (4). The same topology is predicted for the DsrE2A proteins encoded in all members of the Chromatiaceae and Acidithiobacillaceae families listed in ref. 23. Sulfur transferase activity could so far not be proven for this protein. GSSH and thiosulfate cannot serve as sulfur donors for A. vinosum DsrE2A. It is persulfurated by incubation with sulfide in vitro; however, it has not yet been experimentally established whether the active site cysteine is the only residue capable of sulfur binding (4). Just as the DsrE2A protein from A. vinosum, the archaeal protein Mcup_0682 from M. cuprina does not react with GSSH and thiosulfate in vitro (11). The archaeal protein belongs to a different subgroup (DsrE2B) and is an established soluble protein. In contrast to A. vinosum DsrE2A, this protein is not even persulfurated in the presence of sulfide. In summary, the role of DsrE2 proteins remains elusive at present. However, the analysis of M. cuprina DsrE3A (Mcup_681), a protein with a conserved Cys-X7-Cys motif with the active site cysteine as the first residue, provided major insights and suggested a completely new pathway of sulfur input into TusA (Figs. 1 and 3; ref. 11). Both, the archaeal DsrE3A and the TusA protein proved to react with tetrathionate but not with sulfide, GSSH, polysulfide, or thiosulfate. The products were identified as protein-Cys-S-thiosulfonates. MALDI-TOF mass spectrometry confirmed thiosulfate transfer from DsrE3A-thiosulfonate to TusA (11). The back reaction, that is, the transfer of thiosulfate from thiosulfonated TusA to DsrE3A, was not possible. The reaction of tetrathionate with DsrE3A is feasible in vivo. M. cuprina is known to grow on tetrathionate but not on thiosulfate (39) albeit transporters mediating import of tetrathionate into the cytoplasm have not been identified so far (40,41). Altogether, these observations imply that DsrE3A functions as a thiosulfate donor to TusA in vivo. Thiosulfonated TusA could then serve as the substrate for other enzymes such as the Hdr-like system (see below).
The Output Modules Here, the idea is put forward that two principally different output modules exist in sulfur-oxidizing prokaryotes that achieve the oxidation of TusA-bound sulfane sulfur (Fig. 3). The first is the experimentally well-documented delivery of sulfur to reverse-acting dissimilatory sulfite reductase (DsrAB) and the second is the proposed oxidation by an Hdr-like system. The analysis of the sulfur oxidation pathway in A. vinosum revealed major similarities between the E. coli Tus sulfur relay system for tRNA modification and several of the Dsr proteins (5,42). DsrH, DsrF, and DsrE share sequence similarity with TusB, TusC, and TusD, respectively (27). Furthermore, DsrC exhibits sequence similarities with the E. coli protein TusE (27,42). In A. vinosum, DsrEFH and DsrC are encoded in the same gene cluster as DsrAB (18,19). In E. coli, TusA, TusBCD, and TusE act as sulfur transferases and mediate sulfur transfer between the cysteine desulfurase IscS and MnmA, a dedicated
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thiouridylase (27). In a highly organized series of events, the conserved cysteine in the amino-terminus of TusA (Cys19 in the E. coli protein) is first persulfurated by IscS. The persulfide is then transferred to the TusD subunit of TusBCD. Most probably sulfur transfer from TusD to TusE is the next step. Using the persulfide sulfur of TusE, MnmA finally catalyzes ATP-dependent 2thiouridine formation at position 34 of the tRNA (27). Like TusBCD from E. coli, DsrEFH from A. vinosum is a hexameric protein arranged in a a2b2c2 structure (5). It harbors two conserved cysteine residues in the active sites of the DsrE (DsrE-Cys78) and the DsrH (DsrH-Cys20) subunits. Although both of these cysteine residues are essential for sulfur oxidation in vivo, sulfur is only bound to DsrE-Cys78 in vitro. DsrE-Cys78 exactly corresponds to the active site of cysteine residue of E. coli TusD (5). DsrH-Cys20 is not persulfurated on incubation with sulfide (3). DsrEFH on its own is incapable of mobilizing sulfur from persulfidic molecules or thiosulfate (3) and therefore needs a donor protein. A. vinosum TusA is a firmly established interaction partner of DsrEFH. The presence of the conserved active site of cysteine residues in TusA and in the DsrE subunit proved to be essential for the interaction. Sulfur transfer between TusA and DsrEFH is reversible (4). The TusE homolog DsrC is an eminently important 12- to 14-kDa protein that works as the physiological partner of the DsrAB sulfite reductase not only in sulfur-oxidizing prokaryotes but also in sulfate-reducing prokaryotes (23). DsrC is a member of the DsrC/TusE/RpsA superfamily and contains two strictly conserved redox active cysteines in a flexible carboxyterminal arm (42): CysA is the penultimate residue at the Cterminus, and CysB is located about 10 residues upstream. TusE proteins contain only CysA (23). DsrEFH and DsrC from A. vinosum strongly interact in vitro. When combined in solution in their native, nonpersulfurated state, DsrEFH and DsrC form a tight complex as shown by native polyacrylamide gel electrophoresis (3). Each DsrE2F2H2 heterohexamer associates with either one or two DsrC molecules. Interaction of DsrEFH with DsrC is strictly dependent on the presence of DsrE-Cys78 and DsrC-CysA (3,42). Persulfurated DsrEFH transfers sulfur atoms specifically to CysA of A. vinosum DsrC but not to CysB, which was shown by the use of DsrC variants lacking either one or both cysteine residues (3). DsrH-Cys20 is not required for sulfur transfer from DsrEFH to DsrC. The reverse reaction, that is, sulfur transfer from DsrC to DsrEFH was not detected. Persulfurated DsrC serves as the substrate for DsrAB, and the oxidation of DsrC-CysA-S2 by this enzyme is thought to result in persulfonated DsrC (DsrC-CysA-S032) from which sulfite is possibly released by the formation of a disulfide bridge between CysA and CysB (Fig. 1A). The oxidation of sulfite, the product of the DsrAB-catalyzed reaction in sulfur oxidizers, to the final product sulfate is performed either indirectly by APS reductase and ATP sulfurylase via adenosine-50 -phosphosulfate (APS) or directly via the cytoplasmically oriented membrane-bound iron– sulfur molybdoenzyme SoeABC (43).
Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
Recently, a broad database survey revealed a mutually exclusive occurrence of genes encoding Dsr and Hdr-like complex proteins in genome-sequenced sulfur-oxidizing prokaryotes, thus strongly supporting earlier suggestions that the putative Hdr-like complex is involved in a process functionally replacing the Dsr system (22). The immediate linkage of rhd-tusA-dsrE2 or tusA-dsrE2 gene arrangements with the strictly conserved hdrC1B1A1orf2hdrC2B2 gene set in all prokaryotes containing the hdr-like genes in conjunction with the experimentally documented transfer of thiosulfate from M. cuprina DsrE3A to TusA further promotes the notion that an Hdr-like protein complex is involved in the generation of sulfite from TusA-bound sulfur compounds. In species of the genus Acidithiobacillus, organic persulfides that are formed as intermediates during the oxidation of externally available elemental sulfur to sulfite have been suggested as substrates for the sulfur-oxidizing enzymes (44). The Hdr-like system could serve as such a system (10,22,35) and is proposed here to also include TusA as a central element. In archaea like M. cuprina, thiosulfonated TusA could serve as the substrate for the Hdr-like complex (11). In this case, it can be envisioned that the sulfonate group is first released, either hydrolytically as sulfate or reductively as sulfite, by an as yet unknown mechanism. The sulfane group remaining on TusA could then be oxidized and finally also be released (11).
Conclusions In summary, it is evident that protein-mediated sulfur transfer and its delivery to the sulfite-generating enzymes in proteinbound form are of pivotal importance for dissimilatory sulfur oxidation. Extended analyses of arrangements of genes involved in sulfur oxidation confirm not only a tight linkage of (rhd-)tusAdsrE2 with genes encoding major components of the sulfur oxidation machinery in many cases but also indicate a near ubiquitous occurrence of these genes in sulfur oxidizers. The protein TusA appears to be a common element in sulfur oxidizers, and a central role of this protein is proposed. A rhodanese-like protein is needed to mobilize sulfur from lowmolecular-weight organic perthiols and transfers it via TusA (and possibly also via DsrE2), either via DsrEFH and DsrC to DsrAB or directly to the Hdr-like complex (Fig. 3). An alternative route of sulfur input to TusA is exemplified by the archaeal DsrE3A protein that reacts directly with tetrathionate and transfers thiosulfate onto TusA. Finally, DsrAB and alternatively the proposed Hdr-like complex function as the sulfite producing entities. The latter may work only in conjunction with further, so far unidentified, proteins.
Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (Grant Da 351/6-1).
Dahl
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Sulfur Trafficking In Sulfur-Oxidizing Prokaryotes
