Thiosulfate Reductase Research Articles

NADH dehydrogenase is the primary target for the bactericidal effect of CORM-2 in Shewanella oneidensis

Shewanella oneidensis is a good model for investigating the bactericidal effects of chemical agents. In this study, the effects of tricarbonyldichlororuthenium dimer (CORM-2) on the aerobic and anaerobic growth of S. oneidensis were studied, and its bactericidal mechanisms were elucidated. CORM-2 (5 µM) significantly inhibited aerobic and anaerobic respiration. Growth potential tests showed that mutants ΔkatB, ΔoxyR, ΔccmF, and Δcrp and mutants that deleted each cytochrome c-encoding gene exhibited sensitivity to CORM-2 similar to that of the wild-type strain MR-1. Enzymatic analyses showed that cytochrome oxidase, catalase and cysteine desulfurase were activated by CORM-2, while nitrite reductase, nitrate reductase, sulfite reductase, thiosulfate reductase, peroxidase and NADH dehydrogenase were inhibited by CORM-2, and no significant changes were found in the activities of superoxide dismutase and aconitase. Promoter activity assays showed that cco, cyd, cox, katB, sirA and psrA were repressed by CORM-2. Sulfide alleviated the bactericidal effect of CORM-2 and protected NADH dehydrogenase from CORM-2 inhibition. Thus, oxidative stress and cytochrome oxidase inactivation are not the primary reasons for the bactericidal effect of CORM-2, and NADH dehydrogenase is the main target in S. oneidensis. The bactericidal effect of CORM-2 is caused by the toxicity of Ru2+, not by carbon monoxide.

Biodegradation of sulfate and elimination of heavy metals by immobilized-microbial bioaugmentation coupled with anaerobic membrane bioreactor

Sulfate-rich metal-laden wastewater (SRMLW) shows a significant threat on the ecological environment and human health. Sulfate-reducing bacteria (SRB) can remove sulfate from wastewater in anaerobic membrane bioreactor (AnMBR). This study constructed recombinant genetic engineered SRB (rSRB) and investigated the sulfate and heavy metals removal efficiency for treating SRMLW by immobilized-microbial bioaugmentation using rSRB (IMrSRB) coupled with AnMBR (Bio-AnMBR). The results show that Bio-AnMBR removed sulfate (99.4%) from SRMLW by upregulating key genes (Sat, DsrAB, AprAB, QmoABC, DsrC and DsrMKJOP) and enzymes (APS reductase, Dissimilatory sulfite reductase, Trithionate reductase and Thiosulfate reductase) in dissimilatory sulfite and sulfate respiration processes. Bio-AnMBR achieved synergistic biodegradation of sulfate and nitrogen by stimulating the activity of these bacteria related to the conversion of nitrogen and sulfate. Microbial analysis indicated that the relatively high abundance of rSRB (79.2%) and functional genes (70.1%) upregulated the activities of key enzymes associated with sulfate metabolism system, thus increasing the production of S (66.4 mg/L) and H2S (23.8 mg/L). Bio-AnMBR significantly improved the tolerance to heavy metals by increasing concentrations of CO32–, S2-, and HS-, which was configured to reduce heavy metals (95.6%) via precipitation of metal sulfide (81.3 mg/L), metal carbonates (30.7 mg/L) and metal hydroxides (18.7 mg/L). Bio-AnMBR increased system stability, shorted hydraulic retention time (HRT), decreased the production of soluble microbial products (SMP), extracellular polymeric substances (EPS) and waste-sludge production (WSP), as well as improved effluent quality. In conclusion, data in this study indicate that application of Bio-AnMBR is a feasible strategy for the treatment of SRMLW.

A deep-sea sulfate-reducing bacterium generates zero-valent sulfur via metabolizing thiosulfate.

Zero-valent sulfur (ZVS) is a crucial intermediate in the sulfur geobiochemical circulation and is widespread in deep-sea cold seeps. Sulfur-oxidizing bacteria are thought to be the major contributors to the formation of ZVS. However, ZVS production mediated by sulfate-reducing bacteria (SRB) has rarely been reported. In this study, we isolated and cultured a typical SRB designated Oceanidesulfovibrio marinus CS1 from deep-sea cold seep sediment in the South China Sea. We show that O. marinus CS1 forms ZVS in the medium supplemented with thiosulfate. Proteomic and protein activity assays revealed that thiosulfate reductase (PhsA) and the sulfide:quinone oxidoreductase (SQR) played key roles in driving ZVS formation in O. marinus CS1. During this process, thiosulfate firstly was reduced by PhsA to form sulfide, then sulfide was oxidized by SQR to produce ZVS. The expressions of PhsA and SQR were significantly upregulated when O. marinus CS1 was cultured in a deep-sea cold seep, strongly indicating that strain CS1 might form ZVS in the deep-sea environment. Notably, homologs of phsA and sqr were widely identified from microbes living in sediments of deep-sea cold seep in the South China Sea by the metagenomic analysis. We thus propose that SRB containing phsA and sqr genes potentially contribute to the formation of ZVS in deep-sea cold seep environments.

Dietary molybdenum may stimulate the growth of colonic sulfur reducing bacteria, increasing hydrogen sulfide levels in the human colon and the possible health effects of an excess of colonic sulfides

Molybdenum is a trace mineral needed in small quantities by most life forms. In living organisms, a molybdenum atom is found within molybdenum-dependent enzymes or molybdoenzymes. Molybdoenzymes catalyze reactions in carbon, sulfur, and nitrogen metabolism. Only four molbdoenzymes have been identified in humans. Most of the known molybdoenzymes are found in bacteria. Dietary molybdenum can be administrated to humans, to treat Wilson disease and tungsten poisoning; and it may be useful in arthritis. Sulfur-reducing bacteria are the bacterial group that reduces certain sulfur molecules to hydrogen sulfide. These bacteria can inhabit anaerobic parts of the gastrointestinal tract of mammals and are the predominant producer of hydrogen sulfide in the human colon. Hydrogen sulfide plays a major role in the malodor of human flatus. Some individuals have reported an increase in foul odoriferous gases from the colon after molybdenum supplementation. The underlying mechanism as to how this occurs is currently not known. Possible bacteria that are involved could be sulfur-reducing bacteria and methionine dissimilating bacteria. Supplementing sheep with molybdenum and with sulfur exclusively in the form of methionine can stimulate the growth of sulfur-reducing bacteria and increase the level of sulfides in the rumen. The molybdoenzyme, thiosulfate reductase, is found in sulfur-reducing bacteria and catalyzes the reduction of thiosulfate to hydrogen sulfide. The source of thiosulfate could be from ruminal epithelial cells detoxifying methanethiol, produced by methionine dissimilating bacteria, degrading the dietary methionine to methanethiol. Therefore, the molybdenum could be activating thiosulfate reductases of sulfur-reducing bacteria in the rumen of these animals. The human colon can also harbor sulfur-reducing bacteria, and dietary molybdenum and methionine can reach this organ. Therefore, dietary molybdenum may be stimulating the growth of sulfur-reducing bacteria in some individuals. Sulfides in the human colon could have beneficial and detrimental effects on health. Such effects could include the already mentioned malodor of flatus, the stabilizing of the microbiome-mucosa interface in an intestinal dysbiosis, the treatment of hypertension and the promotion of inflammation in ulcerative colitis.

A Cytoplasmic NAD(P)H-Dependent Polysulfide Reductase with Thiosulfate Reductase Activity from the Hyperthermophilic Bacterium Thermotoga maritima.

ABSTRACTThermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments.IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

Thiosulfate oxidation in sulfur-reducing Shewanella oneidensis and its unexpected influences on the cytochrome c content.

Thiosulfate, an important form of sulfur compounds, can serve as both electron donor and acceptor in various microorganisms. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, thiosulfate reduction has long been recognized but whether it can catalyse thiosulfate oxidation remains elusive. In this study, we discovered that S. oneidensis is capable of thiosulfate oxidation, a process specifically catalysed by two periplasmic cytochrome c (cyt c) proteins, TsdA and TsdB, which act as the catalytic subunit and the electron transfer subunit respectively. In the presence of oxygen, oxidation of thiosulfate has priority over reduction. Intriguingly, thiosulfate oxidation negatively regulates the cyt c content in S. oneidensis cells, largely by reducing intracellular levels of cAMP, which as the cofactor modulates activity of global regulator Crp required for transcription of many cyt c genes. This unexpected finding provides an additional dimension to interplays between the respiration regulator and the respiratory pathways in S. oneidensis. Moreover, the data presented here identified S. oneidensis as the first bacterium known to date owning both functional thiosulfate reductase and dehydrogenase, and importantly, genomics analyses suggested that the number of bacterial species possessing this feature is rather limited.

Atypical Non-H2S-Producing Monophasic Salmonella Typhimurium ST3478 Strains from Chicken Meat at Processing Stage Are Adapted to Diverse Stresses.

Poultry products are still an important cause of Salmonella infections worldwide, with an increasingly reported expansion of less-frequent serotypes or atypical strains that are frequently multidrug-resistant. Nevertheless, the ability of Salmonella to survive antimicrobials promoted in the context of antibiotic reducing/replacing and farming rethinking (e.g., organic acids and copper in feed/biocides) has been scarcely explored. We investigated Salmonella occurrence (conventional and molecular assays) among chicken meat at the processing stage (n = 53 batches/29 farms) and characterized their tolerance to diverse stress factors (antibiotics, copper, acid pH, and peracetic acid). Whole-genome sequencing was used to assess adaptive features and to perform comparative analysis. We found a low Salmonella occurrence (4%) and identified S. Enteritidis/ST11 plus atypical non-H2S-producing S. 1,4,[5],12:i:-/ST3478. The ST3478 presented the ability to grow under diverse stresses (antibiotics, copper, and acid-pH). Comparative genomics among ST3478 isolates showed similar antibiotic/metal resistance gene repertoires and identical nonsense phsA thiosulfate reductase mutations (related to H2S-negative phenotype), besides their close phylogenetic relationship by cgMLST and SNPs. This study alerts for the ongoing national and international spread of an emerging monophasic Salmonella Typhimurium clonal lineage with an enlarged ability to survive to antimicrobials/biocides commonly used in poultry production, being unnoticed by conventional Salmonella detection approaches due to an atypical non-H2S-producing phenotype.

Characterization of thiosulfate reductase from Pyrobaculum aerophilum heterologously produced in Pyrococcus furiosus.

The genome of the archaeon Pyrobaculum aerophilum (Topt ~ 100°C) contains an operon (PAE2859-2861) encoding a putative pyranopterin-containing oxidoreductase of unknown function and metal content. These genes (with one gene modified to encode a His-affinity tag) were inserted into the fermentative anaerobic archaeon, Pyrococcus furiosus (Topt ~ 100°C). Dye-linked assays of cytoplasmic extracts from recombinant P. furiosus show that the P. aerophilum enzyme is a thiosulfate reductase (Tsr) and reduces thiosulfate but not polysulfide. The enzyme (Tsr-Mo) from molybdenum-grown cells contains Mo (Mo:W = 9:1) while the enzyme (Tsr-W) from tungsten-grown cells contains mainly W (Mo:W = 1:6). Purified Tsr-Mo has over ten times the activity (Vmax = 1580 vs. 141µmolmin-1 mg-1) and twice the affinity for thiosulfate (Km = ~ 100 vs. ~ 200μM) than Tsr-W and is reduced at a lower potential (Epeak = -255 vs -402mV). Tsr-Mo and Tsr-W proteins are heterodimers lacking the membrane anchor subunit (PAE2861). Recombinant P. furiosus expressing P. aerophilum Tsr could not use thiosulfate as a terminal electron acceptor. P. furiosus contains five pyranopterin-containing enzymes, all of which utilize W. P. aerophilum Tsr-Mo is the first example of an active Mo-containing enzyme produced in P. furiosus.

Engineering bacteria for biogenic synthesis of chalcogenide nanomaterials.

SummaryMicrobes naturally build nanoscale structures, including structures assembled from inorganic materials. Here, we combine the natural capabilities of microbes with engineered genetic control circuits to demonstrate the ability to control biological synthesis of chalcogenide nanomaterials in a heterologous host. We transferred reductase genes from both Shewanella sp. ANA‐3 and Salmonella enterica serovar Typhimurium into a heterologous host (Escherichia coli) and examined the mechanisms that regulate the properties of biogenic nanomaterials. Expression of arsenate reductase genes and thiosulfate reductase genes in E. coli resulted in the synthesis of arsenic sulfide nanomaterials. In addition to processing the starting materials via redox enzymes, cellular components also nucleated the formation of arsenic sulfide nanomaterials. The shape of the nanomaterial was influenced by the bacterial culture, with the synthetic E. coli strain producing nanospheres and conditioned media or cultures of wild‐type Shewanella sp. producing nanofibres. The diameter of these nanofibres also depended on the biological context of synthesis. These results demonstrate the potential for biogenic synthesis of nanomaterials with controlled properties by combining the natural capabilities of wild microbes with the tools from synthetic biology.

Sulfur Redox Cycling Dependent Abiotic Ferrihydrite Reduction by a Desulfitobacterium hafniense

Biogeochemical interactions of iron and sulfur drive their cycles in many environments, which makes understanding the mechanism of sulfur redox cycling dependent abiotic iron(III) reduction by sulfate-reducing bacteria (SRB) particularly important. Here, we present a newly isolated Desulfitobacterium hafniense strain DH with the potential of reducing sulfate, sulfite, thiosulfate, and elemental sulfur from a paddy soil. Strain DH cannot enzymatically reduce ferrihydrite, but it can abiotically reduce ferrihydrite through extracellular electron transfer with biogenic sulfide or other sulfur compounds acting as electron shuttles. Sulfite, elemental sulfur, and thiosulfate, the oxidation products of sulfide, complete the sulfur cycle between ferrihydrite and strain DH. The presence of strain DH at the ferrihydrite surface results in mineral dissolution and secondary mineralization. Proteomic analysis revealed that the expression level of sulfur reduction functional protein DsrC and a thiosulfate reductase in...

Genome Sequence of Desulfurella amilsii Strain TR1 and Comparative Genomics of Desulfurellaceae Family.

The acidotolerant sulfur reducer Desulfurella amilsii was isolated from sediments of Tinto River, an extremely acidic environment. Its ability to grow in a broad range of pH and to tolerate certain heavy metals offers potential for metal recovery processes. Here we report its high-quality draft genome sequence and compare it to the available genome sequences of other members of Desulfurellaceae family: D. acetivorans. D. multipotens, Hippea maritima. H. alviniae, H. medeae, and H. jasoniae. For most species, pairwise comparisons for average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) revealed ANI values from 67.5 to 80% and DDH values from 12.9 to 24.2%. D. acetivorans and D. multipotens, however, surpassed the estimated thresholds of species definition for both DDH (98.6%) and ANI (88.1%). Therefore, they should be merged to a single species. Comparative analysis of Desulfurellaceae genomes revealed different gene content for sulfur respiration between Desulfurella and Hippea species. Sulfur reductase is only encoded in D. amilsii, in which it is suggested to play a role in sulfur respiration, especially at low pH. Polysulfide reductase is only encoded in Hippea species; it is likely that this genus uses polysulfide as electron acceptor. Genes encoding thiosulfate reductase are present in all the genomes, but dissimilatory sulfite reductase is only present in Desulfurella species. Thus, thiosulfate respiration via sulfite is only likely in this genus. Although sulfur disproportionation occurs in Desulfurella species, the molecular mechanism behind this process is not yet understood, hampering a genome prediction. The metabolism of acetate in Desulfurella species can occur via the acetyl-CoA synthetase or via acetate kinase in combination with phosphate acetyltransferase, while in Hippea species, it might occur via the acetate kinase. Large differences in gene sets involved in resistance to acidic conditions were not detected among the genomes. Therefore, the regulation of those genes, or a mechanism not yet known, might be responsible for the unique ability of D. amilsii. This is the first report on comparative genomics of sulfur-reducing bacteria, which is valuable to give insight into this poorly understood metabolism, but of great potential for biotechnological purposes and of environmental significance.

Sulfur-Mediated Electron Shuttling Sustains Microbial Long-Distance Extracellular Electron Transfer with the Aid of Metallic Iron Sulfides.

In addition to serving as an energy source for microbial growth, iron sulfides are proposed to act as naturally occurring electrical wires that mediate long-distance extracellular electron transfer (EET) and bridge spatially discrete redox environments. These hypothetical EET reactions stand on the abilities of microbes to use the interfacial electrochemistry of metallic/semiconductive iron sulfides to maintain metabolisms; however, the mechanisms of these phenomena remain unexplored. To obtain insight into EET to iron sulfides, we monitored EET at the interface between Shewanella oneidensis MR-1 cells and biomineralized iron sulfides in an electrochemical cell. Respiratory current steeply increased with the concomitant formation of poorly crystalline mackinawite (FeS) minerals, indicating that S. oneidensis has the ability to exploit extracellularly formed metallic FeS for long-distance EET. Deletion of major proteins of the metal-reduction (Mtr) pathway (OmcA, MtrC, CymA, and PilD) caused only subtle effects on the EET efficiency, a finding that sharply contrasts the majority of studies that report that the Mtr pathway is indispensable for the reduction of metal oxides and electrodes. The gene expression analyses of polysulfide and thiosulfate reductase suggest the existence of a sulfur-mediated electron-shuttling mechanism by which HS(-) ions and water-soluble polysulfides (HS(n)(-), where n ≥ 2) generated in the periplasmic space deliver electrons from cellular metabolic processes to cell surface-associated FeS. The finding of this Mtr-independent pathway indicates that polysulfide reductases complement the function of outer-membrane cytochromes in EET reactions and, thus, significantly expand the number of microbial species potentially capable of long-distance EET in sulfur-rich anoxic environments.

Systematic genomic analysis reveals the complementary aerobic and anaerobic respiration capacities of the human gut microbiota.

Because of the specific anatomical and physiological properties of the human intestine, a specific oxygen gradient builds up within this organ that influences the intestinal microbiota. The intestinal microbiome has been intensively studied in recent years, and certain respiratory substrates used by gut inhabiting microbes have been shown to play a crucial role in human health. Unfortunately, a systematic analysis has not been previously performed to determine the respiratory capabilities of human gut microbes (HGM). Here, we analyzed the distribution of aerobic and anaerobic respiratory reductases in 254 HGM genomes. In addition to the annotation of known enzymes, we also predicted a novel microaerobic reductase and novel thiosulfate reductase. Based on this comprehensive assessment of respiratory reductases in the HGM, we proposed a number of exchange pathways among different bacteria involved in the reduction of various nitrogen oxides. The results significantly expanded our knowledge of HGM metabolism and interactions in bacterial communities.

Identification of key components in the energy metabolism of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus by transcriptome analyses

Energy conservation via the pathway of dissimilatory sulfate reduction is present in a diverse group of prokaryotes, but is most comprehensively studied in Deltaproteobacteria. In this study, whole-genome microarray analyses were used to provide a model of the energy metabolism of the sulfate-reducing archaeon Archaeoglobus fulgidus, based on comparative analysis of litoautotrophic growth with H2/CO2 and thiosulfate, and heterotrophic growth on lactate with sulfate or thiosulfate. Only 72 genes were expressed differentially between the cultures utilizing sulfate or thiosulfate, whereas 269 genes were affected by a shift in energy source. We identified co-located gene cluster encoding putative lactate dehydrogenases (LDHs; lldD, dld, lldEFG), also present in sulfate-reducing bacteria. These enzymes may take part in energy conservation in A. fulgidus by specifically linking lactate oxidation with APS reduction via the Qmo complex. High transcriptional levels of Fqo confirm an important role of F420H2, as well as a menaquinone-mediated electron transport chain, during heterotrophic growth. A putative periplasmic thiosulfate reductase was identified by specific up-regulation. Also, putative genes for transport of sulfate and sulfite are discussed. We present a model for hydrogen metabolism, based on the probable bifurcation reaction of the Mvh:Hdl hydrogenase, which may inhibit the utilization of Fdred for energy conservation. Energy conservation is probably facilitated via menaquinone to multiple membrane-bound heterodisulfide reductase (Hdr) complexes and the DsrC protein—linking periplasmic hydrogenase (Vht) to the cytoplasmic reduction of sulfite. The ambiguous roles of genes corresponding to fatty acid metabolism induced during growth with H2 are discussed. Putative co-assimilation of organic acids is favored over a homologous secondary carbon fixation pathway, although both mechanisms may contribute to conserve the amount of Fdred needed during autotrophic growth with H2.

Thiosulfate reduction in Salmonella enterica is driven by the proton motive force.

Thiosulfate respiration in Salmonella enterica serovar Typhimurium is catalyzed by the membrane-bound enzyme thiosulfate reductase. Experiments with quinone biosynthesis mutants show that menaquinol is the sole electron donor to thiosulfate reductase. However, the reduction of thiosulfate by menaquinol is highly endergonic under standard conditions (ΔE°' = -328 mV). Thiosulfate reductase activity was found to depend on the proton motive force (PMF) across the cytoplasmic membrane. A structural model for thiosulfate reductase suggests that the PMF drives endergonic electron flow within the enzyme by a reverse loop mechanism. Thiosulfate reductase was able to catalyze the combined oxidation of sulfide and sulfite to thiosulfate in a reverse of the physiological reaction. In contrast to the forward reaction the exergonic thiosulfate-forming reaction was PMF independent. Electron transfer from formate to thiosulfate in whole cells occurs predominantly by intraspecies hydrogen transfer.

Sodium selenate effect on Synechococcus elongatus PCC 7942: appearance of novel enzymatic reaction, ATP:selenate adenylyltransferase, and variation in antioxidant enzyme activities

Synechococcus elongatus PCC 7942 was grown with SO(4) (2-) or S(2)O(3) (2-) sulfur source with varying concentration of SeO(4) (2-). Up to 150 muM, SeO(4) (2-) was growth supportive and above this it was inhibitory. Selenate is believed to induce ROS production, and to counter, the cells produce increasing amounts of ROS quenching enzymes. To investigate the differential growth response of SeO(4) (2-) at the level of ROS production, the activities of antioxidant enzymes viz. glutathione peroxidase, catalase, pyrogallol-peroxidase, superoxide dismutase and glutathione reductase were determined. Regardless of SeO(4) (2-) concentration in the medium, except catalase, the activity of other enzymes increased over SeO(4) (2-)-free controls. To understand the fate of SeO(4) (2-) added at lower concentrations, the activities of key inorganic sulfur metabolizing enzymes, ATP sulfurylase in SO(4) (2-) medium and thiosulfate reductase in S(2)O(3) (2-) medium were measured. This was done with an assumption that these enzymes would consume the SO(4) (2-) analog SeO(4) (2-) as sulfur source. We found enzymatic SeO(4) (2-)/ATP dependent formation of inorganic pyrophosphate in a reaction, analogous to ATP sulfurylase (SO(4) (2-) + ATP --> pyrophosphate) carried out by an enzyme different from ATP sulfurylase, tentatively called ATP:selenate adenylyltransferase. We hypothesize that selenium could act as trace element for S. elongatus PCC 7942.

Anaerobic Respiration of Elemental Sulfur and Thiosulfate by Shewanella oneidensis MR-1 Requires psrA , a Homolog of the phsA Gene of Salmonella enterica Serovar Typhimurium LT2

Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO3(2-)), thiosulfate (S2O3(2-)), tetrathionate (S4O6(2-)), and elemental sulfur (S(0)). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S(0) and S2O3(2-) respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S(0) and S2O3(2-) yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S(0) or S2O3(2-) as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S2O3(2-) terminal reductase whose end products (sulfide [HS-] or SO3(2-)) participate in an intraspecies sulfur cycle that drives S(0) respiration.

Constraints on anaerobic respiration in the hyperthermophilic Archaea Pyrobaculum islandicum and Pyrobaculum aerophilum.

Pyrobaculum islandicum uses iron, thiosulfate, and elemental sulfur for anaerobic respiration, while Pyrobaculum aerophilum uses iron and nitrate; however, the constraints on these processes and their physiological mechanisms for iron and sulfur reduction are not well understood. Growth rates on sulfur compounds are highest at pH 5 to 6 and highly reduced (<-420-mV) conditions, while growth rates on nitrate and iron are highest at pH 7 to 9 and more-oxidized (>-210-mV) conditions. Growth on iron expands the known pH range of growth for both organisms. P. islandicum differs from P. aerophilum in that it requires direct contact with insoluble iron oxide for growth, it did not produce any extracellular compounds when grown on insoluble iron, and it lacked 2,6-anthrahydroquinone disulfonate oxidase activity. Furthermore, iron reduction in P. islandicum appears to be completely independent of c-type cytochromes. Like that in P. aerophilum, NADH-dependent ferric reductase activity in P. islandicum increased significantly in iron-grown cultures relative to that in non-iron-grown cultures. Proteomic analyses showed that there were significant increases in the amounts of a putative membrane-bound thiosulfate reductase in P. islandicum cultures grown on thiosulfate relative to those in cultures grown on iron and elemental sulfur. This is the first evidence of this enzyme being used in either a hyperthermophile or an archaeon. Pyrobaculum arsenaticum and Pyrobaculum calidifontis also grew on Fe(III) citrate and insoluble iron oxide, but only P. arsenaticum could grow on insoluble iron without direct contact.

H2S, a new neuromodulator

The formation of H2S from cyst(e)ine is catalyzed by three enzymes, cystathionine beta synthase, cystathionase, and 3-mercaptopyruvate sulfurtransferase. In the liver, kidney, enterocytes and vascular smooth muscle cells, H2S is principally synthesized by cystathionase. In contrast, it is synthesized by cystathionine beta synthase in the brain and partially by 3-mercaptopyruvate sulfurtransferase in cardiac tissue. H2S is catabolized, essentially in mitochondria by thiosulfate reductase. The sulfite generated is then oxidized to sulfate by sulfite oxidase. The amount of thiosulfate excreted in the urine is the best indicator of H2S biosynthesis, together with sulfhemoglobin determination in erythrocytes. H2S acts as a neuromodulator in the brain, increasing responses mediated by NMDA receptors, facilitating the induction of long-term potentialization in the hippocampus. H2S also acts as a vasodilator, acting directly on ATP-dependent potassium channels in vascular smooth muscle cells. The concentration of H2S is abnormally low in the brains of subjects with Alzheimer's disease, due to changes in the concentration of the physiological activator of cystathionine beta synthase. The overproduction of H2S described in subjects with Down's syndrome probably results from the overproduction of cystathionine beta synthase, as the gene encoding this protein is located on chromosome 21.

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur.

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.