Prokaryotic sulfur oxidation

Prokaryotic sulfur oxidation Cornelius G Friedrich, Frank Bardischewsky, Dagmar Rother, Armin Quentmeier and Jo¨rg Fischer Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the b and g subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiontic bacteria.

Aerobic sulfur oxidation of Archaea is restricted to members of the thermoacidophilic Sulfolobales. In the domain Bacteria, sulfur is oxidized by aerobic chemotrophic [8,9] and anaerobic phototrophic bacteria [10].

Addresses Chair Technical Microbiology, Department of Biochemical and Chemical Engineering, University of Dortmund, D-44221 Dortmund, Germany

In the past, enzymic reactions were the basis to postulate different pathways for sulfur oxidation in different prokaryotes. The characterization of the sulfur oxidizing (Sox) enzyme system of the alphaproteobacterium Paracoccus pantotrophus [14] and the identification of the respective genes in the genomes of other chemotrophic or phototrophic bacteria raised the question of the ‘emergence of a common mechanism’ in bacteria and discriminated the Sox enzyme system from the sulfur-oxidizing proteins in Archaea [8].

Corresponding author: Friedrich, Cornelius G ([email protected])

Current Opinion in Microbiology 2005, 8:253–259 This review comes from a themed issue on Ecology and industrial microbiology Edited by Sergio Sa´nchez and Betty Olson Available online 5th May 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.04.005

Chemolithoautotrophic sulfur bacteria are phylogenetically and physiologically diverse and are alkaliphilic [11,12], neutrophilic or acidophilic [8,9,13]. Also, phototrophic sulfur-oxidizing bacteria are phylogenetically diverse and are mostly mesotrophic and neutrophilic [10,13].

In this review, we use recent data on genomic, biochemical and mutational analysis to describe the sulfuroxidizing enzyme systems of Archaea and Bacteria, and differentiate these in various chemotrophic, phototrophic and acidophilic bacteria.

Sulfur oxidation in the archaeon Acidianus ambivalens Introduction Biological oxidation of hydrogen sulfide or sulfur is abundant in soil and water, and is the major reaction in volcanic and other extreme environments. The oxidation reactions in these ecosystems are performed by prokaryotes of the domains Archaea [1] and Bacteria. Sulfur oxidation by Eukarya is mainly performed by chemotrophic bacterial endosymbionts in worms or mussels of the hydrothermal vent ecosystems [2,3]. Mitochondria of special worms and mussels that survive transient anaerobiosis can detoxify sulfide and this reaction can also be coupled to energy transformation [4–6]. Sulfur occurs in the 2 to +6 oxidation state. The electrons derived from sulfur oxidation are used by aerobic chemotrophic Archaea and Bacteria for energy transformation of the respiratory chain and for autotrophic carbon dioxide reduction. Anaerobic phototrophic bacteria use light energy to transfer electrons from sulfur or other sources for autotrophic carbon dioxide reduction [7]. www.sciencedirect.com

The biological oxidation of reduced inorganic sulfur compounds in extreme environments, such as volcanic hot springs, solfataras and deep-sea hydrothermal vents, is mediated by specialized prokaryotes. Among these, the sulfur-oxidizing enzymes and their genes are best studied in the facultative anaerobic, chemolithoautotrophic, thermoacidophilic archaeon Acidianus ambivalens, a member of the order Sulfolobales. From A. ambivalens several proteins involved in inorganic sulfur metabolism have been described. The cytoplasmic sulfur oxygenase reductase (SOR) catalyzes the conversion of sulfur in the presence of molecular oxygen to give sulfite, thiosulfate and hydrogen sulfide as products, and it was concluded that SOR catalyzes a sulfur disproportionation coupled to an oxygenase reaction [1,15]. SOR is an icosatetrameric enzyme of 871 kDa molecular mass (subunit 35.2 kDa) with a low-potential non-heme iron center [16] for which the crystal structure has been resolved recently [17]. The location of SOR in the cytoplasm and the lack of cofactors, besides iron, do not allow sulfur oxidation to be coupled to Current Opinion in Microbiology 2005, 8:253–259

254 Ecology and industrial microbiology

Figure 1

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Schematic representation of the interaction between thiosulfate:quinone oxidoreductase (TQO) and the A. ambivalens terminal oxygen reductase. The cofactors of the process are copper atoms (Cu), heme a (a), caldariella quinone (CQ), and caldariella quinol (CQH2). The arrow emerging from the terminal oxidase indicates proton pumping. Reproduced from [19] with permission.

electron transport or to substrate level phosphorylation [1]. The membrane-associated SOR of Acidianus tengchongensis contains three cysteine residues within two conserved motifs, V-G-P-K-V-C31 and C101-X-X-C104, and each of these cysteine residues is essential for the catalytic mechanism of SOR. This SOR is co-localized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase [18]. The membrane-bound thiosulfate:quinone oxidoreductase (TQO) has first been described from A. ambivalens. TQO (102 kDa) is composed of 28 kDa and 16 kDa subunits, and oxidizes thiosulfate with tetrathionate as product and ferricyanide or decylubiquinone as electron acceptors. Thiosulfate-dependent oxygen consumption of membranes was measured. The subunits of TQO are identical to DoxDA, which have been described as part of the terminal quinol:oxygen oxidoreductase, thus TQO links thiosulfate consumption to oxygen reduction in a short electron transport (Figure 1) [19].

thiosulfate and are probably involved in transfer of reductants [21]. The subsequent genes soxXYZABCD encode four periplasmic proteins, SoxXA, SoxYZ, SoxB and Sox(CD)2, which reconstitute the Sox enzyme system. SoxXA is a complex composed of the diheme cytochrome c SoxA (30.4 Da) and the monoheme SoxX (14.8 Da). The SoxYZ complex does not contain a metal or cofactor and is composed of SoxY (10.9 Da) and SoxZ (11.7 Da). The invariant Cys138 of the motif (V-K-V-T-I-G-G-C-GG) at the C-terminal end of SoxY covalently binds sulfur of various oxidation states [22]. The SoxYZ complex appears as hetero- and homo-dimers with protein disulfide linked subunits [23] of which the SoxY–Y homodimer might play a role in the reaction cycle. Sox(CD)2 is a a2b2 tetrameric complex of 180 kDa composed of the molybdoprotein SoxC (43.9 Da) and the diheme cytochrome c SoxD (38.8 Da). SoxB (58.6 Da) contains a dinuclear manganese cluster [24], is proposed to function as sulfate thiohydrolase and interacts with the SoxYZ complex [23]. The reconstituted Sox enzyme system of P. pantotrophus catalyzes the thiosulfate-dependent reduction of horse cytochrome c, and it reacts with hydrogen sulfide and sulfur at higher rate than it reacts with thiosulfate, and with sulfite at a lower rate than with thiosulfate. This substrate versatility is remarkable as these sulfur compounds are neither isosteric nor isoelectronic, and differ in redox-potential and reactivity [14]. The current model [8] proposes SoxXA to oxidatively link thiosulfate to the sulfhydryl of Cys138 of SoxY to yield thiocysteine-S-sulfate. The crystal structure has been resolved from SoxXA of the phototrophic nonsulfur purple bacterium Rhodovulum sulfidophilum giving the structural basis of its function as a heme enzyme [25,26]. Spectroscopic studies identified heme-1 of SoxA of R. sulfidophilum not to be redox-reactive [27], and heme-1 is completely missing from SoxA of Starkeya novella [28]. Thiocysteine-S-sulfate of SoxY is the proposed substrate for SoxB to yield cysteine-persulfide and sulfate. The outer sulfur atom of cysteine-persulfide is oxidized to cysteine-S-sulfate by Sox(CD)2. Finally, sulfate is again hydrolyzed off by SoxB to regenerate SoxY [8].

The sox gene cluster The genes encoding sulfur-oxidizing (Sox) ability were first described from the alphaproteobacterium P. pantotrophus, which is a facultative chemolithoautotroph and grows with thiosulfate. The sox gene cluster comprises 15 genes (Figure 2). soxR encodes a repressor protein of the ArsR family SoxR, which binds to the soxS–V and soxW–X intergenic regions. The binding is not affected by thiosulfate or sulfide and is possibly redox-directed. SoxS, a periplasmic thioredoxin, is essential for full expression of the sox genes [20]. The soxVW and subsequent sox genes are divergently transcribed to soxRS. soxV encodes a membrane protein and soxW encodes a periplasmic thioredoxin. Both are essential for chemotrophic growth with Current Opinion in Microbiology 2005, 8:253–259

The soxF gene encodes the monomeric flavoprotein SoxF that has sulfide dehydrogenase activity [29]. A novel activity has been discovered for SoxF to activate the thiosulfate- or sulfide-oxidizing Sox enzyme system when reconstituted with a SoxYZ protein separately inactivated by reduction. Thus, SoxF has a redox-balancing function that very probably reflects its function in vivo (CG Friedrich et al., unpublished).

The significance of sulfur dehydrogenase SoxCD The Sox enzyme system yields eight mol electrons per mol of thiosulfate whereas only two mol electrons are www.sciencedirect.com

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Figure 2

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Schematic overview of the sox-locus of P. pantotrophus and related genes of chemotrophic and phototrophic sulfur-oxidizing bacteria. Genes encoding homologous proteins are indicated by identical colors. The localization of the genes on the chromosomes is given by the nucleotide numbering. Completely sequenced genomes are indicated by black numbers and partially sequenced chromosomes are indicated by red numbers. The number of the DNA contigs is given in brackets. The following databases were used for sequence comparison: DOE JGI database (C. metallidurans, Magnetococcus MC-1, M. magnetotacticum); Genome Information Broker database (T. thermophilus, R. solanacearum, C. tepidum, A. aeolicus). Data for A. vinosum were communicated by C Dahl. Sequence information for the other strains was taken from the EMBL database.

yielded when Sox(CD)2 is omitted from the reconstituted enzyme system. Thus, Sox(CD)2 mediates a unique oxidative six electron transfer, represents a novel type of molybdenum enzyme, and was designated sulfur dehydrogenase [8]. The yield of two electrons from thiosulfate by the Sox system lacking Sox(CD)2 suggests that sulfur or polysulfide is the product of this reaction. The chemotrophic sulfur bacterium S. novella oxidizes sulfite, whereas P. pantotrophus does not because of the lack of sulfite dehydrogenase. Sulfite oxidase from S. novella is a heterodimeric complex of a molybdoprotein and a c-type cytochrome encoded by the sorAB genes, which are located separately from the sox gene cluster (Figure 2). S. novella harbors the soxCD genes for sulfur dehydrogenase within the sox gene cluster [30,31]. Although sulfite is oxidized by the Sox enzyme system of P. pantotrophus in vitro, sulfur dehydrogenase is not www.sciencedirect.com

required for this reaction. The characteristics of sulfite oxidase of prokaryotic sources [30] differ from sulfur dehydrogenase with respect to the catalytic reaction, structure and function [8]. Sulfur dehydrogenase is complexed with a special cytochrome that carries the heme-1 domain of SoxD of P. pantotrophus, SoxD1 (CG Friedrich and co-workers, unpublished; see update). The heme-2 domain is present in SoxD of the chemolithoheterotrophic bacterium Silicibacter pomeroyi [32] and the chemotroph Pseudaminobacter salicylatoxidans but is missing from SoxD of other chemotrpophic and phototrophic sulfur-oxidizing bacteria (Figure 3). SoxD1 contains a novel motif, P-C-M-X-(A/N)-C, which possibly indicates its function as heme enzyme. The heme-2 domain is not required for catalytic activity and electron yield of the sulfur dehydrogenase complex but determines the tetrameric structure as evident from a mutant of P. pantotrophus lacking the complete heme-2 domain of SoxD, and Current Opinion in Microbiology 2005, 8:253–259

256 Ecology and industrial microbiology

Figure 3

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Paracoccus pantotrophus Silicibacter pomeroyi Rhodovulum sulphidophilum Starkeya novella Pseudaminobacter salicylatoxidans Methylobacterium extorquens Bradyrhizobium japonicum Rhodopseudomonas palustris Current Opinion in Microbiology

Schematic representation of the cytochrome SoxD polypeptide of various bacteria. Note the constant distance between the heme-binding motif, C-X-X-C-H, and the novel SoxD1-specific motif P-C-M-X-(D/N)-C. The second heme-binding motif of P. pantotrophus, S. pomeroyi and P. salicylatoxidans is located on the cytochrome c2 domain of the hybrid cytochromes.

which forms a heterodimeric SoxCD1 complex of 90 kDa (CG Friedrich and co-workers, unpublished; see update). The bacterial endosymbiont of the marine deep-sea tubeworm Riftia pachyptila is supplied by its host with sulfide. Sulfide is oxidized to sulfite by a system that involves electron transport, cytochromes, adenylphosphosulfate reductase and ATP sulfurylase (see [2,3] and references therein).

Sulfur oxidation of phototrophic bacteria The Chlorobiaceae are anoxygenic phototrophic green sulfur bacteria that oxidize hydrogen sulfide to sulfuric acid and transiently deposit sulfur globules outside the cell. Carbon dioxide is fixed autotrophically by way of the reductive tricarboxylic acid cycle [33]. The genome of Chlorobium tepidum, a moderate thermophile, contains a cluster of 13 genes of which soxFXYZAB are homologous to the respective genes of P. pantotrophus [34] (Figure 2). The involvement of the sox genes in the sulfur metabolism of C. tepidum is evident from a pleiotropic mutant, strain V::RLP, which lacks the Rubisco-like protein (RLP) and is defective in thiosulfate oxidation but is able to oxidize sulfide just as the wild-type strain does [33]. Thus, the incomplete Sox enzyme system is functional in thiosulfate oxidation and can release sulfur (or polysulfide) from the Sox system. The absence of the soxCD genes from the C. tepidum genome suggests that this bacterium oxidizes sulfur to sulfate via a different reaction. Instead, C. tepidum contains duplicated dsr gene clusters encoding dissimilatory siroheme sulfite reductase and other proteins. Also, Allochromatium vinosum harbors the dsr cluster of which several genes were characterized [35,36]. The anoxygenic phototrophic purple sulfur bacterium A. vinosum is a gammaproteobacterium. From A. vinosum the soxAXB and soxYZ genes have been identified. The small gene clusters are located at two different positions in the Current Opinion in Microbiology 2005, 8:253–259

chromosome (C Dahl, personal communication). A. vinosum transiently deposits protein-coated globules of sulfur in the periplasm as an obligate intermediate during sulfide and thiosulfate oxidation to sulfate. Stored sulfur is present as sulfur chains, probably as organylsulfanes (RSn-R or R-Sn-H where n  4) [37,38].

The dsr gene cluster The involvement of the dsr genes in mobilization of sulfur deposits for anaerobic sulfur oxidation of A. vinosum have been identified by the pioneering work of Dahl and co-workers [35] who established a genetic system and demonstrated by insertional mutagenesis the crucial role of the dsr gene cluster for oxidation of intracellular sulfur. The complete dsr gene cluster of A. vinosum comprises fifteen genes, dsrABEFHCMKLJOPNRS [36]. The ubiquitous presence of dsr genes in anoxygenic phototrophic sulfur bacteria stresses their importance in sulfur oxidation. The dsrAB genes encode sulfite reductase, a protein that is closely related to dissimilatory sulfite reductases from sulfate-reducing bacteria and Archaea. DsrKJO is purified from membranes pointing at the presence of a transmembrane electron-transporting complex consisting of DsrKMJOP, which copurifies with DsrAB (sulfite reductase) [36]. Insertional mutagenesis of dsr genes proved that these genes are essential for oxidation of stored sulfur globules. However, these mutants are unaffected in their ability to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic and under photoorganoheterotrophic growth conditions [35]. It is, therefore, a pending question how sulfur is finally oxidized to sulfate in A. vinosum. The betaproteobacterium Thiobacillus denitrificans is an obligately lithoautotrophic, facultative anaerobic sulfuroxidizing bacterium, which transiently deposits sulfur. This bacterium differs from all other bacteria in its ability to grow anaerobically with thiosulfate as an electron donor www.sciencedirect.com

Prokaryotic sulfur oxidation Friedrich et al. 257

Figure 4

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Schematic overview of the dsr locus of A. vinosum and related genes of T. denitrificans and C. tepidum. Genes encoding homologous proteins are indicated by identical colors or letters. The sequences were obtained for T. denitrificans from DOE JGI database, for C. tepidum from Genome Information Broker database and for A. vinosum from GenBank, accession number U84760. The genes of the dsr gene cluster of A. vinosum and C. tepidum were annotated according to Dahl et al. [36].

and with nitrate and other oxidized nitrogen compounds as electron acceptors, which are metabolized to sulfate and dinitrogen. Over two decades ago, Schedel and Tru¨ per proposed that siroheme sulfite reductase acts in the reverse direction as the crucial enzyme for sulfur oxidation owing to the differences of enzyme characteristics compared with assimilatory enzymes, reviewed in [13]. Analysis of the genome sequence of T. denitrificans, which is to date incomplete, uncovered an almost identical dsr gene cluster to that in A. vinosum [36] (J Fischer, unpublished). The dsr gene cluster is duplicated in C. tepidum (Figure 4). A. vinosum and C. tepidum harbour sox genes in addition to the dsr cluster. Also, from T. denitrificans soxXA genes have been identified predicting periplasmic cytochromes. Sequence analysis suggested a periplasmic SoxB-like protein, whereas the soxYZ genes were found to be missing from the as yet incomplete genome sequence (J Fischer, unpublished). It should be noted that A. vinosum — just like as T. denitrificans — is capable of switching from anaerobic to aerobic sulfur oxidation. A. vinosum, like other purple sulfur bacteria, grows with thiosulate chemotrophically in the dark, albeit under reduced oxygen partial pressure. This is reviewed in [13]. The presence of basic sox genes (soxXABYZ) and their essentiality for sulfur oxidation in C. tepidum suggested that their combination with the dsr gene cluster enabled the phototrophic and — by analogy — chemotrophic bacteria to oxidize thiosulfate and sulfide to sulfate, possibly involving sulfite dehydrogenase. www.sciencedirect.com

Genome analysis of sulfur-oxidizing prokaryotes Genes homologous to the sor gene of the archaeon A. ambivalens have been found in the genomes of the Archaea A. tengchongensis (formerly Acidianus strain S5 [39]), Sulfolobus tokodaii and Ferroplasma acidarmanus and in the hyperthermophilic bacterium Aquifex aeolicus with identities ranging from 39% (A. aeolicus) to 88% (A. tengchongensis). No similar gene was found from the genome of the mesophilic acidophile Acidithiobacillus ferrooxidans [16]. However, A. ferrooxidans harbors duplicated doxDA genes that are homologous to the genes encoding TQO in A. ambivalens. This gene duplication points to a yet undemonstrated significance in thiosulfate metabolism of this acidophilic bacterium. Also, genes closely related to the doxDA genes are present in S. solfataricus and S. tokodaii, whereas those found in the genome sequences of two Bacteroides species are distantly related [19]. Genes homologous to those encoding Sox proteins of P. pantotrophus have been identified from partially and completely sequenced genomes of members of the domain Bacteria but not in the domain Archaea [8]. The gammaproteobacterium A. ferrooxidans does not harbor sox genes stressing the significance of its doxDA genes. Complete sox gene clusters encoding essential components of the Sox enzyme system in P. pantotrophus are present in partially sequenced genomes of chemotrophic bacteria such as S. novella, Methylobacterium extorquens, Pseudaminobacter salicylatoxidans and Bradyrhizobium japonicum. The latter has not been shown to oxidize reduced Current Opinion in Microbiology 2005, 8:253–259

258 Ecology and industrial microbiology

inorganic sulfur compounds. Also, complete sox gene clusters are present in the phototrophs R. sulfidophilum, Rhodopseudomonas palustris and the chemolithoheterotroph Silicibacter pomeroyi (Figure 2). Incomplete sox gene clusters without soxCD genes, which encode sulfur dehydrogenase essential for chemotrophic growth of P. pantotrophus, are present in the genomes of several bacteria: the thermophilic chemotroph Aquifex aeolicus and the mesophiles Ralstonia solanacearum, Cupriavidus metallidurans (Ralstonia metallidurans [40]), and Magnetococcus MC-1 for which the chemotrophic phenotype have not been established. The soxCD genes are also missing from the sox gene cluster of the phototrophic bacteria Chlorobium limicola [41] and C. tepidum (Figure 2).

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Conclusions The understanding of the mechanism of sulfur oxidation in the thermoacidophilic archaeon A. ambivalens has made considerable progress with the discovery of TQO activity and its genes, and their identification in A. ferrooxidans. The Sox enzyme system of the alphaproteobacterium P. pantotrophus is so far the best described system with respect to the proteins involved and their partial reactions. Beta- and gamma-proteobacteria and chlorobia harbor a sox gene cluster without soxCD and the phenotype of a C. tepidum mutant suggests the involvement of the partial sox cluster in sulfur oxidation. The common characteristic of C. tepidum and of A. vinosum is the formation of sulfur globules, the absence of soxCD genes from the sox cluster and the presence of the dsr genes. The chemotrophic bacteria that miss soxCD do not deposit sulfur and do not harbor dsr genes. Therefore, it remains a pressing question with which system these bacteria oxidize sulfur to sulfate.

Update The study cited in the text as CG Friedrich and coworkers, unpublished, has now been accepted for publication [42].

Acknowledgments We thank Christiane Dahl for making available the sox sequence data of A. vinosum prior to publication. The financial support of the Deutsche Forschungsgemeinschaft to CGF (grant Fr318/8-1 and Fr318/9-1) is gratefully acknowledged.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest Kletzin A, Urich T, Mu¨ ller F, Bandeiras TM, Gomes CM: Dissimilatory oxidation and reduction of elemental sulfur in thermophilic bacteria. J Bioenerg Biomembr 2004, 36:77-91. This review summarizes the state of the art of the enzymes involved in sulfur oxidation of Acidianus ambivalens and their significance in sulfur disproportionation, and aerobic and anaerobic energy transformation.

1. 

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Current Opinion in Microbiology 2005, 8:253–259