Intermediates of denitrification in the chemoautotroph Thiobacillus denitrificans

Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO₃ ^-, NO, and N₂O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - NHQNO 2-n-nonyl-4-hydroxyquinoline N-oxide     

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H₂, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O₂ as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O₂ concentrations or ceased after repeated O₂ additions. The amounts of O₂ consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O₂ completely to CO₂. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O₂ probably required a reductive first step, since it was obtained only with energized intact cells.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - APS adenosine phosphosulfate or adenylyl sulfate     

Detoxification of environmental sulfide to sulfane sulfur in the intertidal sipunculid Phascolosoma arcuatum

The capability of Phascolosoma arcuatum to detoxify sulfide in anaerobic conditions was examined. Sulfane sulfur, which underwent cold cyanolysis, was the major excretory end product of sulfide detoxification during anoxia. Thiosulfate was not excreted into the external medium. Instead, it was absorbed by P. arcuatum and its absorption was stimulated by the presence of sodium sulfide (Na₂S) in the incubation medium. The effective formation and excretion of sulfane sulfur by P.␣arcuatum required the presence of both Na₂S and sodium thiosulfate (Na₂S₂O₃). Results obtained indicate that rhodanese might be involved in sulfide detoxification in this sipunculid. Rhodanese could act as a catalyst in the transfer of sulfur atoms from thiosulfate to HS⁻. The body wall and the introvert were the main sites of sulfide detoxification. However, it is unlikely that epibiotic bacteria associated with the outside surface of the worm were involved in the detoxification process. A time-course study on the contents of thiosulfate and sulfane sulfur in the body wall of P. arcuatum incubated anaerobically in the presence of Na₂S + Na₂S₂O₃ verified that thiosulfate absorbed was utilized to detoxify sulfide to sulfane sulfur. Accepted: 24 October 1996     

The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis

Purple sulfur bacterium Thiocapsa roseopersicina strain BBS requiring vitamin B₁₂ may grow in the dark in media containing no other organic compounds. Under such conditions the cells oxidize sulfide and thiosulfate with the use of O₂ and assimilate carbon dioxide. After 10–30 s assimilation of NaH¹⁴CO₃ about 60% of radioactivity is found in phosphorylated compounds characteristic for the reductive pentose phosphate cycle. The possibility of the function of this cycle in the dark in the presence of O₂ is confirmed by the capacity of cells grown under such conditions to synthesize ribulose-1,5-diphosphate carboxylase. All this evidence suggests the ability of T. roseopersicina to change from phototrophy to aerobic chemolithoautotrophy.     

Reduced sulfur and nitrogen compounds and molecular hydrogen as electron donors for anaerobic CO2 photoreduction in Anacystis nidulans

Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O₂ evolution and CO₂ fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O₂ evolution much more strongly than CO₂ fixation, indicating ability to supply electrons for CO₂ photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO₂ fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO₂ fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O₂. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H₂ and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone - DCMU dichlorophenyl dimethyl urea - DSPD disalicylidenepropane diamine-(1,3) - EDAC 1-ethyl-3(3-dimethylaminopropyl-) carbodiimide     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

biochemical reaction mechanisms in sulfur oxidation by chemosynthetic bacteria

Summary Aspects of the biochemistry of the oxidation of inorganic sulfur compounds are discussed in thiobacilli but chiefly inThiobacillus denitrificans. Almost all of the thiobacilli (e.g. T. denitrificans, T. neapolitanus, T. novellus, andThiobacillus A ₂) were capable of producing approximately 7.5 moles of sulfuric acid aerobically from 3.75 moles of thiosulfate per gram of cellular protein per hr. By far the most prolific producer of sulfuric acid (or sulfates) from the anaerobic thiosulfate oxidation with nitrates wasT. denitrificans which was capable of producing 15 moles of sulfates from 7.5 moles of thiosulfate with concomitant reduction of 12 moles of nitrate resulting in the evolution of 6 moles of nitrogen gas/g protein/hr. The oxidation of sulfide was mediated by the flavo-protein system and cytochromes ofb, c, o, anda-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. The aerobic thiosulfate oxidation on the other hand involved cytochromec : O₂ oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate byT. denitrificans, however, was severely inhibited by the flavoprotein inhibitors because of the splitting of the thiosulfate molecule into the sulfide and sulfite moieties produced by the thiosulfate-reductase. Accumulation of tetrathionate and to a small extent trithionate and pentathionate occurred during anaerobic growth ofT. denitrificans. These polythionates were subsequently oxidized to sulfate with the concomitant reduction of nitrate to N₂. Intact cell suspensions catalyzed the complete oxidation of sulfide, thiosulfate, tetrathionate, and sulfite to sulfate with the stoichiometric reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas thus indicating that NO₂ ⁻, NO, and N₂O are the possible intermediates in the denitrification of nitrate. This process was mediated by the cytochrome electron transport chain and was sensitive to the electron transfer inhibitors. The oxidation of sulfite involved cytochrome-linked sulfite oxidase as well as the APS-reductase pathways. The latter was absent inT. novellus andThiobacillus A ₂. In all of the thiobacilli the inner as well as the outer sulfur atoms of thiosulfate were oxidized at approximately the same rate by intact cells. The sulfide oxidation occurred in two stages: (a) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (b) a slower oxidation reaction stage catalyzed by the cellfree extracts, probably involving polysulfides. InT. novellus andT. neapolitanus the oxidation of inorganic sulfur compounds is coupled to energy generation through oxidative phosphorylation, however, the reduction of pyridine nucleotides by sulfur compounds involved an energy-linked reversal of electron transfer. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974. Summary already inserted on p. 189 of the present volume.     

Energy transduction efficiencies in nitrogenous oxide respirations of Azospirillum brasilense Sp7

For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H⁺-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth yields (Y _s ^max -values) were essentially the same with O₂ or N₂O but were 1/3 and 2/3 lower with NO ₂ ^- or NO ₃ ^- , respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N₂-fixing conditions drastically reduced the Y _s ^max -values in the N₂O and O₂-respiring cells. In the H⁺-translocation measurements, the /oxidant ratios were 5.6 for O₂→H₂O, 2.5–2.8 for NO ₃ ^- →NO ₂ ^- , 2.2 for NO ₂ ^- →N₂O and 3.1 for N₂O→N₂ respirations when the cells were preincubated with valinomycin and K⁺. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP⁺) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H⁺-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore, nitrate and nitrite respirations were accompanied by a H⁺-uptake . Any significant H⁺-translocation could not be detected in N₂O- and O₂-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal molar growth yield determinations and the absence of any H⁺-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of respiration. The low H⁺-extrusion values for N₂O-respiration compared to O₂-respiration in cells treated with valinomycin plus TPMP⁺ are, however, not in accord with such an interpretation.     

Lithoautotrophic Growth of the Freshwater Colorless Sulfur Bacterium Beggiatoa “leptomitiformis”D-402

The freshwater colorless sulfur bacterium Beggiatoa leptomitiformisD-402 was shown to be capable of lithoautotrophic growth in a batch culture under microoxic conditions at O₂concentrations in the medium of no higher than 0.5 mg/l. The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l. A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells. The high rate of CO₂assimilation (112–139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91–92% of which was accounted for by CO₂carbon, were close to those typical of autotrophic bacteria. Thiosulfate was oxidized almost completely to sulfate, and the fraction of intracellular sulfur in the final products did not exceed 0.2–1.7% of the thiosulfate sulfur. The cell membrane fraction contained cytochromes (b + o) and two cytochromes cwith M _rof 23 and 26 kDa; the soluble fraction contained cytochrome cwith M _rof 12 kDa.     

Thiosulfate production from cystine by the keratinolytic prokaryote Streptomyces fradiae

In cultures of Streptomyces fradiae on wool as the only source of nutrition inorganic thiosulfate (in amounts up to 0.5 mg of Na₂S₂O₃·5 H₂O/ml) was formed as the final product of metabolization of sulfur from cystine of keratin proteins. The presence of thiosulfate was proved by qualitative tests and thin-layer chromatography and estimated quantitatively by spectrophotometry, titrimetry, and capillary isotachophoresis. Metabolization of organic sulfur to thiosulfate excreted into the medium is a process not yet described in microorganisms.     

Oxidation of dimethylsulfide to tetrathionate by Methylophaga thiooxidans sp. nov.: a new link in the sulfur cycle

A new pathway of dimethylsulfide (DMS) metabolism was identified in a novel species of Gammaproteobacteria, Methylophaga thiooxidans sp. nov., in which tetrathionate (S₄O₆^2?) was the end‐product of DMS oxidation. Inhibitor evidence indicated that DMS degradation was initiated by demethylation, catalysed by a corrinoid demethylase. Thiosulfate was an intermediate, which was oxidized to tetrathionate by a cytochrome‐linked thiosulfate dehydrogenase. Thiosulfate oxidation was coupled to ATP synthesis, and M. thiooxidans could also use exogenous thiosulfate as an energy source during chemolithoheterotrophic growth on DMS or methanol. Cultures grown on a variety of substrates oxidized thiosulfate, indicating that thiosulfate oxidation was constitutive. The observations have relevance to interactions among sulfur‐metabolizing bacteria in the marine environment. The production of tetrathionate from an organosulfur precursor is previously undocumented and represents a potential step in the biogeochemical sulfur cycle, providing a ‘shunt’ across the cycle.     

Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing Xanthobacter tagetidis

Xanthobacter tagetidis grew as a chemolithotrophic autotroph on thiosulfate and other inorganic sulfur compounds, as a heterotroph on thiophene-2-carboxylic acid, acetic acid and α-ketoglutaric acid, and as a mixotroph on thiosulfate in combination with thiophene-2-carboxylic acid and/or acetic acid. Autotrophic growth on one-carbon organosulfur compounds, and intermediates in their oxidation are also reported. Thiosulfate enhanced the growth yields in mixotrophic cultures, presumably by acting as a supplementary energy source, since ribulose bisphosphate carboxylase was only active in thiosulfate-grown cells and was not detected in mixotrophic cultures using thiosulfate with thiophene-2-carboxylic acid. Bacteria grown on thiophene-2-carboxylic acid also oxidized sulfide, thiosulfate and tetrathionate, indicating these as possible sulfur intermediates in thiophene-2-carboxylic acid degradation. Thiosulfate and tetrathionate were oxidized completely to sulfate and, consequently, did not accumulate as products of thiophene-2-carboxylic acid oxidation in growing cultures. K _m and V _max values for the oxidation of thiosulfate, tetrathionate or sulfide were 13 μM and 83 nmol O₂ min^–1 (mg dry wt.)^–1, respectively; thiosulfate and tetrathionate became autoinhibitory at concentrations above 100 μM. The true growth yield (Y_max) on thiophene-2-carboxylic acid was estimated from chemostat cultures (at dilution rates of 0.034–0.094 h^–1) to be 112.2 g mol^–1, with a maintenance coefficient (m) of 0.3 mmol thiophene-2-carboxylic acid (g dry wt.)^–1 h^–1, and the maximum specific growth rate (μ_max) was 0.116 h^–1. Growth in chemostat culture at a dilution rate of 0.041 h^–1 indicated growth yields [g dry wt. (mol substrate)^–1] of 8.1 g (mol thiosulfate)^–1, 60.9 g (mol thiophene-2-carboxylic acid)^–1, and 17.5 g (mol acetic acid)^–1, with additive yields for growth on mixtures of these substrates. At a dilution rate of 0.034 h^–1, yields of 57.8 g (mol α-ketoglutaric acid)^–1 and 60.7 g (mol thiophene-2-carboxylic acid)^–1 indicated some additional energy conservation from oxidation of the thiophene-sulfur. SDS-PAGE of cell-free preparations indicated a polypeptide (M _r, 21.0 kDa) specific to growth on thiophene-2-carboxylic acid for which no function can yet be ascribed: no metabolism of thiophene-2-carboxylic acid by cell-free extracts was detected. It was shown that X. tagetidis exhibits a remarkable degree of metabolic versatility and is representative of facultatively methylotrophic and chemolithotrophic autotrophs that contribute significantly to the turnover of simple inorganic and organic sulfur compounds (including substituted thiophenes) in the natural environment. Received: 1 July 1997 / Accepted: 3 November 1997     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Capacity of Azospirillum thiophilum for lithotrophic growth coupled to oxidation of reduced sulfur compounds

Capacity for lithotrophic growth coupled to oxidation of reduced sulfur compounds was revealed in an Azospirillum strain, A. thiophilum BV-S ^T . Oxygen concentration in the medium was the major factor determining the type of energy metabolism (organotrophic or lithotrophic) in the presence of thiosulfate. Under aerobic conditions, metabolism of A. thiophilum BV-S^T was organoheterotrophic, with thiosulfate oxidation to tetrathionate resulting from the interaction with reactive oxygen species, mostly H₂O₂, which was formed in the electron transport chain in the course of oxidation of organic electron donors. Under microaerobic conditions (2 mg/L O₂ in liquid medium), A. thiophilum BV-S^T carried out lithoheterotrophic (mixotrophic) metabolism; enzymes of the dissimilatory type of sulfur metabolism were responsible for thiosulfate oxidation to tetrathionate and sulfate. Two enzyme systems were found in the cells: thiosulfate dehydrogenase, which catalyzes incomplete oxidation of thiosulfate to tetrathionate and the thiosulfate-oxidizing Sox enzyme complex, which is involved in complete oxidation of thiosulfate to sulfate. The genetic determinant of a Sox complex component in A. thiophilum BV-S^T was revealed. The soxB gene was found, and its expression under microaerobic conditions was observed to increase 32-fold compared to aerobic cultivation.     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Capacity of chromatiaceae for chemotrophic growth. Specific respiration rates of Thiocystis violacea and Chromatium vinosum

The capacity for chemoautotrophic, mixotrophic and organotrophic growth in the dark was tested with 45 strains of 17 species (11 genera) of the Chromatiaceae. The auxanographic deep agar shake culture method was used; the gas phase contained 5% O₂ and 1% CO₂ in N₂. All strains tested of Chromatium vinosum, C. minus, C. violascens, C. gracile, Thiocystis violacea, Amoebobacter roseus, Thiocapsa roseopersicina gave positive growth responses under chemoautotrophic and mixotrophic conditions (extra carbon source acetate); one strain of Thiocapsa roseopersicina grew also organotrophically on acetate alone. No growth was obtained with the remaining 17 strains of ten species. None of the five type species (three genera) of the Chlorobiaceae grew under chemotrophic conditions. With Thiocystis violacea 2311 a growth yield of 11.3g dry weight per mol thiosulfate consumed was obtained under chemoautotrophic conditions; under mixotrophic conditions with acetate the yield increased to 69g dry weight per mol thiosulfate consumed. With Thiocystis violacea 2311 maximal specific respiration rates were obtained with thiosulfate as electron donor irrespective of the presence or absence of sulfur globules in the cells; organic substrates served as carbon sources only and did not support respiration. With Chromatium vinosum D utilization of thiosulfate was not constitutive; maximal respiration rates on thiosulfate were obtained only with thiosulfate grown cells containing sulfur globules. Respiration rates were further increased by malate, fumarate or propionate; these substrates also served as sole electron donors for respiration. Acetate and pyruvate were used as carbon sources only. The ecological significance of the chemotrophic metabolism is discussed.     

The functional role of reduced inorganic sulfur compounds in the metabolism of the microaerophilic bacterium <Emphasis Type="Italic">Spirillum winogradskii</Emphasis>

Oxidation of reduced sulfur compounds by the microaerophilic sulfur bacterium spirillum winogradskii was found to occur only concomitantly with consumption of an organic substrate and was not linked to their utilization as electron donors in energy metabolism. No enzymes of dissimilatory sulfur metabolism were found in the cells of the sulfur bacterium oxidizing thiosulfate to tetrathionate; oxidation of thiosulfate and sulfide was caused by their reaction with reactive oxygen species (ROSs), mostly H₂O₂ produced in the course of aerobic growth. A decreased lytic effect of ROSs in the presence of thiosulfate resulted in a twofold increase in the cell yield under aerobic conditions and more efficient substrate utilization. The latter effect was caused by decreased expenditure of energy for the biosynthesis of oxygen-protective polysaccharides. The stimulatory effect of thiosulfate on the growth processes was due to the activation of a number of TCA cycle enzymes producing the intermediates for constructive metabolism, especially of the NADP-dependent malic enzyme. As a result of thiosulfate-induced synthesis of SH-containing cell components, the integral antioxidative activity increased 1.5-fold.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 17–25.Original Russian Text Copyright © 2005 by Podkopaeva, Grabovich, Dubinina.     

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.     

<Emphasis Type="Italic">Thiocapsa imhoffii</Emphasis>, sp. nov., an alkaliphilic purple sulfur bacterium of the family <Emphasis Type="Italic">Chromatiaceae</Emphasis> from Soap Lake,Washington (USA)

An alkaliphilic purple sulfur bacterium, strain SC5, was isolated from Soap Lake, a soda lake located in east central Washington state (USA). Cells of strain SC5 were gram-negative, non-motile, and non-gas vesiculate cocci, often observed in pairs or tetrads. In the presence of sulfide, elemental sulfur was deposited internally. Liquid cultures were pink to rose red in color. Cells contained bacteriochlorophyll a and spirilloxanthin as major photosynthetic pigments. Internal photosynthetic membranes were of the vesicular type. Optimal growth of strain SC5 occurred in the absence of NaCl (range 0–4%), pH 8.5 (range pH 7.5–9.5), and 32°C. Photoheterotrophic growth occurred in the presence of sulfide or thiosulfate with only a limited number of organic carbon sources. Growth factors were not required, and cells could fix N₂. Dark, microaerobic growth occurred in the presence of both an organic carbon source and thiosulfate. Sulfide and thiosulfate served as electron donors for photoautotrophy, which required elevated levels of CO₂. Phylogenetic analysis placed strain SC5 basal to the clade of the genus Thiocapsa in the family Chromatiaceae with a 96.7% sequence similarity to its closest relative, Thiocapsa roseopersicina strain 1711^T (DSM217^T). The unique assemblage of physiological and phylogenetic properties of strain SC5 defines it as a new species of the genus Thiocapsa, and we describe strain SC5 herein as Tca. imhoffii, sp. nov.     

Growth kinetics of haloalkaliphilic,sulfur-oxidizing bacterium<Emphasis Type="Italic"> Thioalkalivibrio versutus</Emphasis> strain ALJ 15 in continuous culture

The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (K_s=6±3 M). The maintenance requirement (m_s=8±5 mmol S₂O₃²/g dry weight h^–1) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (S_n^2–) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.Communicated by W.D. Grant