Identification of MET10-932 and characterization as an allele reducing hydrogen sulfide formation in wine strains of Saccharomyces cerevisiae

A vineyard isolate of the yeast Saccharomyces cerevisiae, UCD932, was identified as a strain producing little or no detectable hydrogen sulfide during wine fermentation. Genetic analysis revealed that this trait segregated as a single genetic determinant. The gene also conferred a white colony phenotype on BiGGY agar (bismuth-glucose-glycine-yeast agar), which is thought to indicate low basal levels of sulfite reductase activity. However, this isolate does not display a requirement for S-containing amino acids, indicating that the sulfate reduction pathway is fully operational. Genetic crosses against known mutations conferring white colony color on BiGGY agar identified the gene leading to reduced H(2)S formation as an allele of MET10 (MET10-932), which encodes a catalytic subunit of sulfite reductase. Sequence analysis of MET10-932 revealed several corresponding amino acid differences in relation to laboratory strain S288C. Allele differences for other genes of the sulfate reduction pathway were also detected in UCD932. The MET10 allele of UCD932 was found to be unique in comparison to the sequences of several other vineyard isolates with differing levels of production of H(2)S. Replacing the MET10 allele of high-H(2)S-producing strains with MET10-932 prevented H(2)S formation by those strains. A single mutative change, corresponding to T662K, in MET10-932 resulted in a loss of H(2)S production. The role of site 662 in sulfide reduction was further analyzed by changing the encoded amino acid at this position. A change back to threonine or to the conservative serine fully restored the H(2)S formation conferred by this allele. In addition to T662K, arginine, tryptophan, and glutamic acid substitutions similarly reduced sulfide formation.     

Identification of Genes Affecting Hydrogen Sulfide Formation in Saccharomyces cerevisiae

A screen of the Saccharomyces cerevisiae deletion strain set was performed to identify genes affecting hydrogen sulfide (H₂S) production. Mutants were screened using two assays: colony color on BiGGY agar, which detects the basal level of sulfite reductase activity, and production of H₂S in a synthetic juice medium using lead acetate detection of free sulfide in the headspace. A total of 88 mutants produced darker colony colors than the parental strain, and 4 produced colonies significantly lighter in color. There was no correlation between the appearance of a dark colony color on BiGGY agar and H₂S production in synthetic juice media. Sixteen null mutations were identified as leading to the production of increased levels of H₂S in synthetic juice using the headspace analysis assay. All 16 mutants also produced H₂S in actual juices. Five of these genes encode proteins involved in sulfur containing amino acid or precursor biosynthesis and are directly associated with the sulfate assimilation pathway. The remaining genes encode proteins involved in a variety of cellular activities, including cell membrane integrity, cell energy regulation and balance, or other metabolic functions. The levels of hydrogen sulfide production of each of the 16 strains varied in response to nutritional conditions. In most cases, creation of multiple deletions of the 16 mutations in the same strain did not lead to a further increase in H₂S production, instead often resulting in decreased levels.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

MET17 and Hydrogen Sulfide Formation in Saccharomyces cerevisiae

Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H₂S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H₂S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H₂S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H₂S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H₂S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H₂S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H₂S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H₂S production and release.     

Polymorphic Variants of Human Rhodanese Exhibit Differences in Thermal Stability and Sulfur Transfer Kinetics

Rhodanese is a component of the mitochondrial H₂S oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306A→C, has an allelic frequency of 1% and results in an E102D substitution in the encoded protein. The second polymorphism, 853C→G, has an allelic frequency of 5% and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfur transfer reactions. The Asp-102 and Ala-285 variants are more stable than wild-type rhodanese and exhibit k_cat/K_m,CN values that are 17- and 1.6-fold higher, respectively. All three rhodanese forms preferentially catalyze sulfur transfer from GSSH to sulfite, generating thiosulfate and glutathione. The k_cat/K_m,sulfite values for the variants in the sulfur transfer reaction from GSSH to sulfite were 1.6- (Asp-102) and 4-fold (Ala-285) lower than for wild-type rhodanese, whereas the k_cat/K_m,GSSH values were similar for all three enzymes. Thiosulfate-dependent H₂S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.     

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Characterization of a Thermolabile Sulfite Reductase from Salmonella pullorum

The biochemical basis for sulfite accumulation by sulfate-using revertants of Salmonella pullorum was determined. All of the sulfate-using mutants isolated from wild-type S. pullorum accumulated sulfite when grown at 37 but not at 25 C. The specific activity of reduced nicotinamide adenine dinucleotide (NADPH)-dependent sulfite reductase (H ₂S-NADP oxidoreductase, EC 1.8.1.2) and of reduced methyl viologen (MVH)-dependent sulfite reductase (H ₂S-MV oxidoreductase), in extracts prepared from cells incubated at 37 C, declined as the incubation period lengthened. However, the specific activity of both reductases from cells incubated at 25 C did not decline. Thermolability of cell-free NADPH-dependent sulfite reductase from cells of S. pullorum incubated at 37 C was greater than the lability of this enzyme either from cells of S. typhimurium incubated at 37 C or from cells of S. pullorum incubated at 25 C. Cells cultured at 37 C continued to accumulate sulfite when the incubation temperature was shifted to 25 C; cells cultured at 25 C and shifted to 37 C accumulated no sulfite, whereas these cells shifted to 41 C accumulated sulfite. It was concluded that the configuration of the sulfite reductase of S. pullorum strain 6–18 is a function of the incubation temperature at which synthesis occurs.     

Hydrogen sulfide-producing kinetics of Shewanella oneidensis in sulfite and thiosulfate respiration

Shewanella oneidensis is a model species for aquatic ecosystems and plays an important role in bioremediation, biofuel cell manufacturing and biogeochemical cycling. S. oneidensis MR-1 is able to generate hydrogen sulfide from various sulfur species; however, its catalytic kinetics have not been determined. In this study, five in-frame deletion mutants of S. oneidensis were constructed and their H₂S-producing activities were analyzed. SirA and PsrA were the two major contributors to H₂S generation under anoxic cultivation, and the optimum SO₃²⁻ concentration for sulfite respiration was approximately 0.8 mM, while the optimum S₂O₃²⁻ concentration for thiosulfate respiration was approximately 0.4 mM. Sulfite and thiosulfate were observed to interfere with each other during respiration, and a high concentration of sulfite or thiosulfate chelated extracellular free-iron but did not repress the expression of sirA or psrA. Nitrite and nitrate were two preferred electron acceptors during anaerobic respiration; however, under energy-insufficient conditions, S. oneidensis could utilize multiple electron acceptors simultaneously. Elucidiating the stoichiometry of H₂S production in S. oneidensis would be helpful for the application of this species in bioremediation and biofuel cell manufacturing, and would help to characterize the ecophysiology of sulfur cycling.     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

Evidence for an intracellular sulfur cycle in cucumber leaves

H₂S emission from cucumber (Cucumis sativus L.) leaf discs supplied with L-cysteine in the dark is inhibited 80–90% by aminooxyacetic acid (AOA), an inhibitor of pyridoxal-phosphate dependent enzymes. Exposure to L-cysteine in the light enhanced the emission of H₂S in response to this sulfur source. Turning off the light reduced the emission of H₂S to the rate observed in continuous dark; turning on the light enhanced the emission of H₂S to the rate observed in continuous light. Therefore, in the light H₂S emission in response to L-cysteine becomes a partially light-dependent process. Treatment with cyanazine, an inhibitor of photosynthetic electron transport, reduced H₂S emission in the light to the rate observed in continuous dark, but did not affect H₂S emission in the dark. In leaf discs pre-exposed to L-cysteine in the light, treatment with cyanazine+ AOA inhibited the emission of H₂S in response to L-cysteine completely. Therefore, only part of the H₂S emitted in response to this sulfur source is derived from a light-independent, but pyridoxal-phosphate-dependent process; the balance of the H₂S emitted is derived from a light-dependent process that can be inhibited by cyanazine. When cucumber leaf discs were supplied with a pulse of L-[^35S]cysteine, radioactively labeled H₂S was emitted in two waves, one during the first hour of exposure to L-cysteine, and a second after 3–4 h; unlabeled H₂S, however, was emitted continuously. The second wave of emission of labeled H₂S was not observed in pulse-chase experiments in which sulfate or cyanazine were added to the treatment solution after 3 h of exposure to L-cysteine, or when the lights was turned off. The labeling pattern of sulfur compounds inside cucumber cells supplied with a pulse of L-[³⁵S]cysteine showed that the labeled H₂S released from L-cysteine partially enters first the sulfite, then the sulfate pool of the cells. The radioactively labeled sulfate, however, is not incorporated into L-cysteine, but enters the H₂S pool of the cells again. These observations are consistent with the idea of an intracellular sulfur cycle in plant cells. The L-cysteine taken up by the leaf discs seems to be desulfhydrated in a light-independent, but pyridoxal-phosphate-dependent process. The H₂S synthesized this way may be partially released into the atmosphere; the other part of the H₂S produced in response to L-cysteine may be oxidized to sulfite, then to sulfate, which is subsequently reduced via the light-depent sulfate assimilation pathway. In the presence of excess L-cysteine, synthesis of additional cysteine may be inhibited, and the sulfide moiety may be split off carrier bound sulfide to enter the H₂S pool of the cells again. It is suggested that the function of this sulfur cycle may be regulation of the free cysteine pool.Abbreviation AOA aminooxyacetic acid     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution‐based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease–deficient variants that unable to produce sulfites and hydrogen sulfide (H₂S). Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty‐four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO₂ <10 mg l^?1) and with an impaired H₂S production in grape must without added sulfites were selected. Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. Significance and Impact of the Study: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H₂S.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.     

Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

A malic Acid permease in isolated vacuoles of a crassulacean Acid metabolism plant

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO₂ in the field emit H₂S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H₂S is the only reduced sulfurous compound emitted from SO₂ fumigated leaves. The emission is light and SO₂ concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H₂S several hours after the termination of prolonged SO₂ fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.     

Reduction of Sulfur Compounds in the Sediments of a Eutrophic Lake Basin

Concentrations of various sulfur compounds (SO₄²⁻, H₂S, S⁰, acid-volatile sulfide, and total sulfur) were determined in the profundal sediments and overlying water column of a shallow eutrophic lake. Low concentrations of sulfate relative to those of acid-volatile sulfide and total sulfur and a decrease in total sulfur with sediment depth implied that the contribution of dissimilatory sulfur reduction to H₂S production was relatively minor. Addition of 1.0 mM Na₂³⁵SO₄ to upper sediments in laboratory experiments resulted in the production of H₂³⁵S with no apparent lag. Kinetic experiments with ³⁵S demonstrated an apparent K_m of 0.068 mmol of SO₄²⁻ reduced per liter of sediment per day, whereas tracer experiments with ³⁵S indicated an average turnover time of the sediment sulfate pool of 1.5 h. Total sulfate reduction in a sediment depth profile to 15 cm was 15.3 mmol of sulfate reduced per m² per day, which corresponds to a mineralization of 30% of the particulate organic matter entering the sediment. Reduction of ³⁵S⁰ occurred at a slower rate. These results demonstrated that high rates of sulfate reduction occur in these sediments despite low concentrations of oxidized inorganic compounds and that this reduction can be important in the anaerobic mineralization of organic carbon.     

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αβγ)₂ of about 390 kDa. The K_M for sulfite and k_cat values were 34 μM and 567 s⁻¹ respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ₁₀ analogue, the decyl-ubiquinone, as well, with a K_M of 2.6 μM and a k_cat of 52.9 s⁻¹. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.     

Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

A key step in sulfate assimilation into cysteine is the reduction of sulfite to sulfide by sulfite reductase (SiR). This enzyme is encoded by three genes in the moss Physcomitrella patens. To obtain a first insight into the roles of the individual isoforms, we deleted the gene encoding the SiR1 isoform in P. patens by homologous recombination and subsequently analysed the ΔSiR1 mutants. While ΔSiR1 mutants showed no obvious alteration in sulfur metabolism, their regeneration from protoplasts and their ability to produce mature spores was significantly affected, highlighting an unexpected link between moss sulfate assimilation and development, that is yet to be characterized.     

Light-dependent Emission of Hydrogen Sulfide from Plants

With the aid of a sulfur-specific flame photometric detector, an emission of volatile sulfur was detected from leaves of cucumber (Cucumis sativus L.), squash and pumpkin (Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine max [L.] Merr.) and cotton (Gossypium hirsutum L.). The emission was studied in detail in squash and pumpkin. It occurred following treatment of the roots of plants with sulfate and was markedly higher from either detached leaves treated via the cut petiole, or whole plants treated via mechanically injured roots. Bisulfite elicited higher rates of emission than sulfate. The emission was completely light-dependent and increased with light intensity. The rate of emission rose to a maximum and then declined steadily toward zero in the course of a few hours. However, emission resumed after reinjury of roots, an increase in light intensity, an increase in sulfur anion concentration, or a dark period of several hours.

The emission was identified as H₂S by the following criteria: it had the odor of H₂S; it was not trapped by distilled H₂O, but was trapped by acidic CdCl₂ resulting in the formation of a yellow precipitate, CdS; it was also trapped by base and the contents of the trap formed methylene blue when reacted with N,N-dimethyl-p-phenylenediamine and Fe³⁺.

H₂S emission is not the cause of leaf injury by SO₂, since bisulfite produced SO₂ injury symptoms in dim light when H₂S emission was low, while sulfate did not produce injury symptoms in bright light when H₂S emission was high.

The maximum rates of emission observed, about 8 nmol min⁻¹ g fresh weight⁻¹, are about the activity that would be expected for the sulfur assimilation pathway of a normal leaf. H₂S emission may be a means by which the plant can rid itself of excess inorganic sulfur when HS⁻ acceptors are not available in sufficient quantity.