Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer <Emphasis Type="Italic">Allochromatium vinosum</Emphasis>

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.     

Expression of genes for sulfur oxidation in the intracellular chemoautotrophic symbiont of the deep-sea bivalve Calyptogena okutanii

To understand sulfur oxidation in thioautotrophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox) (soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.     

Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Sulfate‐reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS‐reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone‐interacting membrane‐bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native‐PAGE complexome profiling and 2D BN‐/SDS‐PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN‐/SDS‐PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na⁺‐based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese‐like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.     

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180^T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum. Received: 5 November 1997 / Accepted: 30 March 1998     

DsrL mediates electron transfer between NADH and rDsrAB in Allochromatium vinosum

Dissimilatory sulphite reductase DsrAB occurs in sulphate/sulphite-reducing prokaryotes, in sulphur disproportionators and also in sulphur oxidizers, where it functions in reverse. Predictions of physiological traits in metagenomic studies relying on the presence of dsrAB, other dsr genes or combinations thereof suffer from the lack of information on crucial Dsr proteins. The iron–sulphur flavoprotein DsrL is an example of this group. It has a documented essential function during sulphur oxidation and was recently also found in some metagenomes of probable sulphate and sulphite reducers. Here, we show that DsrL and reverse acting rDsrAB can form a complex and are copurified from the phototrophic sulphur oxidizer Allochromatium vinosum. Recombinant DsrL exhibits NAD(P)H:acceptor oxidoreductase activity with a strong preference for NADH over NADPH. In vitro, the rDsrABL complex effectively catalyses NADH-dependent sulphite reduction, which is strongly enhanced by the sulphur-binding protein DsrC. Our work reveals NAD⁺ as suitable in vivo electron acceptor for sulphur oxidation in organisms operating the rDsr pathway and points to reduced nicotinamide adenine dinucleotides as electron donors for sulphite reduction in sulphate/sulphite-reducing prokaryotes that contain DsrL. In addition, dsrL cannot be used as a marker distinguishing sulphate/sulphite reducers and sulphur oxidizers in metagenomic studies without further analysis.     

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an ₄₄-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×10³ cm²xmmol^-1 at 392 nm, 98×10³ cm² xmmol^-1 at 595 nm and 22×10³ cm²x-mmol^-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e^-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.Non-common abbreviations APS adenylyl sulfate - SDS sodium dodecyl sulfate     

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180^T). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling σ⁷⁰-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which – when compared to the isolated proteins – carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Δp that drives ATP synthesis and reverse electron flow in Chr. vinosum. Received: 1 October 1997 / Accepted: 17 December 1997     

Coexistence of Chlorobium and Chromatium in a sulfide-limited continuous culture

Competition experiments between Chromatium vinosum and Chlorobium limicola in sulfide-limited continuous culture under photolithoautotrophic conditions resulted in the coexistence of both organisms. The ratio between the two bacteria was dilution-rate as well as pH dependent. The observed coexistence can be explained as a hitherto not reported form of dual substrate limitation. The two substrates involved are the electron donors sulfide (growth-limiting substrate in the reservoir vessel) and extracellular elemental sulfur (formed by Chlorobium as a result of sulfide oxidation). It is argued that, although Chlorobium may have the better affinity for both substrates involved, Chromatium can compete successfully on the basis of its intracellular storage of sulfur. Ecological implication of the observed coexistence with respect to natural blooms are discussed.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

Inorganic sulfur oxidizing system in green sulfur bacteria

Green sulfur bacteria use various reduced sulfur compounds such as sulfide, elemental sulfur, and thiosulfate as electron donors for photoautotrophic growth. This article briefly summarizes what is known about the inorganic sulfur oxidizing systems of these bacteria with emphasis on the biochemical aspects. Enzymes that oxidize sulfide in green sulfur bacteria are membrane-bound sulfide-quinone oxidoreductase, periplasmic (sometimes membrane-bound) flavocytochrome c sulfide dehydrogenase, and monomeric flavocytochrome c (SoxF). Some green sulfur bacteria oxidize thiosulfate by the multienzyme system called either the TOMES (thiosulfate oxidizing multi-enzyme system) or Sox (sulfur oxidizing system) composed of the three periplasmic proteins: SoxB, SoxYZ, and SoxAXK with a soluble small molecule cytochrome c as the electron acceptor. The oxidation of sulfide and thiosulfate by these enzymes in vitro is assumed to yield two electrons and result in the transfer of a sulfur atom to persulfides, which are subsequently transformed to elemental sulfur. The elemental sulfur is temporarily stored in the form of globules attached to the extracellular surface of the outer membranes. The oxidation pathway of elemental sulfur to sulfate is currently unclear, although the participation of several proteins including those of the dissimilatory sulfite reductase system etc. is suggested from comparative genomic analyses.     

The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR

During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules. The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC. Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A. vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules. Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes. The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions. Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the main proteins of the sulfur globule envelope. Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules. This indicates a role of SgpC for globule expansion. A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.The paper is dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel, Göttingen, on the occasion of his 80th birthday on October 24th, 2004, with great gratitude, as our interest in microbial sulfur metabolism goes back to the early 1960s, when HGT worked in Prof. Schlegels laboratory and in 1972 established this field in Bonn.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. The proteins associated with sulfur globules of Chromatium vinosum and Thiocapsa roseopersicina were isolated by extraction into 50% aqueous acetonitrile containing 1% trifluoroacetic acid and 10 mM dithiothreitol. The extracted proteins were separated by reversed-phase HPLC, revealing three major proteins from C. vinosum and two from T. roseopersicina. All of these proteins have similar, rather unusual amino acid compositions, being rich in glycine and aromatic amino acids, particularly tyrosine. The molecular masses of the C. vinosum proteins were determined to be 10,498, 10,651, and 8,479 Da, while those from T. roseopersicina were found to be 10,661 and 8,759 Da by laser desorption time-of-flight mass spectrometry. The larger T. roseopersicina protein is N-terminally blocked, probably by acetylation, but small amounts of the unblocked form (mass = 10,619) were also isolated by HPLC. Protein sequencing showed that the two larger C. vinosum proteins are homologous to each other and to the large T. roseopersicina protein. The 8,479 Da C. vinosum and 8,759 Da T. roseopersicina proteins are also homologous, indicating that sulfur globule proteins are conserved between different species of purple sulfur bacteria.Abbreviations BNPS-skatole 2 (2-Nitrophenylsulfenyl)-3-methyl-3-bromoindolenine - CNB Cyanogen bromide - Cv1, Cv2, and Cv3 Chromatium vinosum sulfur globule proteins - SGP and SGPs Sulfur globule protein(s) - TFA Trifluoroacetic acid - Tr0, Tr1, and Tr2 Thiocapsa roseopersicina sulfur globule proteins     

Sulfur metabolism in Beggiatoa alba

The metabolism of sulfide, sulfur, and acetate by Beggiatoa alba was investigated under oxic and anoxic conditions. B. alba oxidized acetate to carbon dioxide with the stoichiometric reduction of oxygen to water. In vivo acetate oxidation was suppressed by sulfide and by several classic respiratory inhibitors, including dibromothymoquinone, an inhibitor specific for ubiquinones. B. alba also carried out an oxygen-dependent conversion of sulfide to sulfur, a reaction that was inhibited by several electron transport inhibitors but not by dibromothymoquinone, indicating that the electrons released from sulfide oxidation were shuttled to oxygen without the involvement of ubiquinones. Intracellular sulfur stored by B. alba was not oxidized to sulfate or converted to an external soluble form under aerobic conditions. On the other hand, sulfur stored by filaments of Thiothrix nivea was oxidized to extracellular soluble oxidation products, including sulfate. Sulfur stored by filaments of B. alba, however, was reduced to sulfide under short-term anoxic conditions. This anaerobic reduction of sulfur was linked to the endogenous oxidation of stored carbon and to hydrogen oxidation.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid