Nitrate as a sole nitrogen source forMethanococcus thermolithotrophicus and its effect on growth of several methanogenic bacteria

Methanococcus (Mc.) thermolithotrophicus can use nitrate as the sole source of nitrogen, but four other species of methanogens cannot. The growth rate was similar on both nitrate and ammonium, but yields were 20–25% lower on nitrate.Mc. thermolithotrophicus, Methanobacterium thermoautotrophicum, andMethanobrevibacterium smithii were not inhibited by 20 mM nitrate, butMethanospirillum hungatei was inhibited 35%, andMethanosarcina barkeri was completely inhibited by 20 mM nitrate. WhenMc. thermolithotrophicus was growing with nitrate as the sole source of nitrogen, growth was dependent on either molybdenum or tungsten, and the presence of both gave the best growth response; vanadium or chromium did not replace the requirement for these metals. Growth on ammonium could not be strictly demonstrated to require either of these metals, but both molybdenum and tungsten stimulated growth.     

Continuous chemotrophic growth and respiration of Chromatiaceae species at low oxygen concentrations

Endogenous and maximum respiration rates of nine purple sulfur bacterial strains were determined. Endogenous rates were below 10 nmol O₂ · (mg protein · min)^-1 for sulfur-free cells and 15–35 nmol O₂ · (mg protein · min)^-1 for cells containg intracellular sulfur globules. With sulfide as electron-donating substrate respiration rates were considerably higher than with thiosulfate. Maximum respiration rates of Thiocystis violacea 2711 and Thiorhodovibrio winogradskyi SSP1 (254.8 and 264.2 nmol O₂ · (mg protein · min)^-1, respectively) are similar to those of aerobic bacteria. Biphasic respiration curves were obtained for sulfur-free cells of Thiocystis violacea 2711 and Chromatium vinosum 2811. In Thiocystis violacea the rapid and incomplete oxidation of thiosulfate was five times faster than the oxidation of stored sulfur. A high affinity of the respiratoty system for oxygen (K _m =0.3–0.9 M O₂, V _max=260 nmol O₂ · (mg protein · min)^-1 with sulfide as substrate, K _m =0.6–2.4 M O₂, V _max=14–40 nmol O₂ · (mg protein · min)^-1 with thiosulfate as substrate), for sulfide (K _m =0.47 M, V _max=650 nmol H₂S · (mg protein × min)^-1, and for thiosulfate (K _m =5–6 M, V _max =24–72 nmol S₂O ₃ ^2- · (mg protein · min)^-1 was obtained for different strains. Respiration of Thiocystis violacea was inhibited by very low concentrations of NaCN (K _i =1.7 M) while CO concentrations of up to 300 M were not inhibitory. The capacity for chemotrophic growth of six species was studied in continuous culture at oxygen concentrations of 11 to 67 M. Thiocystis violacea 2711, Amoebobacter roseus 6611, Thiocapsa roseopersicina 6311 and Thiorhodovibrio winogradskyi SSP1 were able to grow chemotrophically with thiosulfate/acetate or sulfide/acetate. Chromatium vinosum 2811 and Amoebobacter purpureus ML1 failed to grow under these conditions. During shift from phototrophic to chemotrophic conditions intracellular sulfur and carbohydrate accumulated transiently inside the cells. During chemotrophic growth bacteriochlorophyll a was below the detection limit.     

Production of elemental sulfur by green and purple sulfur bacteria

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

Interpretation of sulfur cycling in two catchments in the Black Forest (Germany) using stable sulfur and oxygen isotope data

The isotopic composition of SO ₄ ^2- in bulk precipitation, canopy throughfall, seepage water at three different soil depths, stream water, and groundwater was monitored in two forested catchments in the Black Forest (Germany) between November 1989 and February 1992. Isotope measurements on aqueous sulfate were complemented by ³⁴S-analyses on SO₂ in the air, total sulfur and inorganic sulfate in the soil, and bedrock sulfur, in order to identify sources and biogeochemical processes affecting S cycling in catchments with base poor, siliceous bedrock. Stable S isotope data indicated that atmospheric deposition and not mineral weathering is the major source of S in both catchments since ³⁴S-values for sulfate in the soil, in seepage water, and in stream water were generally found to be similar to the mean ³⁴S-values of precipitation SO ₄ ^2- (+2.1. However, ¹⁸O-values of seepage water SO ₄ ^2- at 30 cm and especially at 80 cm depth were depleted by several per mil with respect to those of the atmospheric deposition (+7.5 to +13.5. This indicates that in both catchments a considerable proportion of the seepage water SO ₄ ^2- is derived from mineralization of carbon-bonded soil S and must therefore have cycled through the organic soil S pool. ³⁴S-values for different S compounds in the solid soil were found to differ markedly depending on S fraction and soil depth. Since atmospheric S deposition with rather constant ³⁴S-values was identified as the dominant S source in both catchments, this is interpreted as a result ofin situ isotope fractionation rather than admixture of isotopically different S. The differences between the ³⁴S-values of seepage water and soil sulfate and those of organic soil S compounds are consistent with a model in which SO ₄ ^2- uptake by vegetation and soil microorganisms favours³⁴SO ₄ ^2- slightly, whereas during mineralization of organic soil S to aqueous SOSO ₄ ^2- ,³²S reacts preferentially. However, the data provide evidence for negligible isotope fractionation during physico-chemical S transformations such as adsorption/desorption in aerated forest soils.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

The use of stable sulfur isotope labelling to elucidate sulfur metabolism by Clostridium pasteurianum

An unique stable isotope labelling experiment was conducted whereby mixtures of sulfate and sulfite of different isotopic compositions were metabolized by Clostridium pasteurianum. The results showed during reduction of 1 mM SO ₃ ⁼ plus 1 mM SO ₄ ⁼ , essentially all evolved H₂S arose from the sulfite whereas in the case of cellular sulfur, 85% was derived from sulfite and the remainder from sulfate.     

Isotope exchange reactions with radiolabeled sulfur compounds in anoxic seawater

The isotope exchange between³⁵S-labeled sulfur compounds of sulfate (SO₄ ^2–), elemental sulfur (S⁰), polysulfide (S_n ^2–), hydrogen sulfide (HS^–: H₂S + HS^– + S^2–), iron sulfide (FeS), and pyrite (FeS₂) was studied at pH 7.6 and 20 °C in anoxic, sterile seawater. Isotope exchange was observed between S⁰, S₂ ^2– HS^–, and FeS, but not between³⁵S labeled SO₄ ^2– or FeS₂ and the other sulfur compounds. Polysulfide mediated the isotope exchange between S⁰ and bisulfide (HS^–). The isotope exchange between S⁰ and S_n ^2–) reached 50% of equilibrium within < 2 min while exchange between S₂ ^2– and HS^– approached equilibrium within 0.5-1 h. In all the experiments HS^–, revealed a fraction exchange from 0.79 to 1.00. Isotope exchange between S^2– and FeS took place only via S₂ ^2– and/or HS^–. The isotope exchange between iron sulfide and the other sulfur compounds was not complete within 24 h as shown by a fraction exchange of 0.07–0.83. This lack of equilibrium (fraction exchange < 1) was due to the isotope exchange between dissolved compounds and surfaces of sulfur particles. The isotopic exchange reactions limit the usefulness of radiotracers in process studies of the inorganic sulfur species. Exchange reactions will also affect the stable isotope distribution among the sulfur species. The kinetics of the isotopic exchange reactions, however, depend on both pH and temperature.     

Dibenzothiophene sulfur can serve as the sole electron acceptor during growth by sulfate-reducing bacteria

Summary Three species of Sulfate-Reducing Bacteria (SRB) were able to grow using dibenzothiophene (DBT) as their sole source of sulfur and sole electron acceptor. Desulfotomaculum orientis and Desulfovibrio desulfuricans were grown at 30°C while Thermodesulfobacterium commune was grown at 60°C, in media containing lactate and citrate. Hydrogen sulfide was the product of dissimilatory sulfur reduction.Research supported by the Office of Oil and Gas Processing of Fossil Energy, U.S. Department of Energy under contract DE-AC05-84OR21400 with Martin Marietta Energy Systems Inc.     

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification,characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A₂₈₀/A₄₁₆=0.39) shows maxima at 551 nm (-band), 521 nm (-band), and 416 nm (-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A₂₈₀/A₄₁₇=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A₂₈₀/A₄₁₈=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.The soluble fraction further contains the non-heme ironcontaining proteins rubredoxin and ferredoxin. The anaerobic sulfide oxidation in a growing culture of Chlorobium vibrioforme f. thiosulfatophilum is accompanied by a rapid formation of thiosulfate, which is only utilized when sulfide is no longer available, while the elemental sulfur concentration increases constantly until thiosulfate is consumed.Non-common abbreviations C Chlorobium - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein     

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.     

Utilization of sulfide and elemental sulfur by Ectothiorhodospira halochloris

Ectothiorhodospira halochloris grows photoheterotrophically with a variety of sulfur sources. During sulfide oxidation to elemental sulfur considerable amounts of polysulfides may be accumulated transiently. When grown on elemental sulfur no sulfate was produced by oxidation, but sulfide and polysulfide were formed by reduction. Only one soluble cytochrome c-551 was isolated and purified. It was a small acidic hemeprotein with a molecular weight of 6,300, an isoelectric point of 3.1 and a redox potential of-11 mV at pH 7.0. It showed three absorption maxima in the reduced state (=551 nm; =523 nm; =417 nm). The addition of various c-type cytochromes to a suspension of spheroplasts stimulated the velocity of sulfide oxidation. This stimulation was best with the small acidic cytochromes from E. halochloris or Ectothiorhodospira abdelmalekii. Sulfide oxidation was stopped by several uncoupling agents, ionophores and electron transport inhibitors. Antimycin A, rotenone and cyanide had no effect on sulfide oxidation.Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Cytochromes,rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (-band at 553 nm, -band at 523 nm, -band at 417 nm). The best purity index (A ₂₈₀/A ₄₁₇) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A ₂₈₀/A ₄₁₈=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (-band), 523 nm (-band) and 418 nm (-band), while the oxidized form has the -band at 413 nm.Rubredoxin (best purity index obtained: A ₂₈₀/A ₄₉₀=3.5) is an acidic small protein. Its molecular weight estimated by gel filtration and SDS acrylamide gel electrophoresis is 27,000 and 6,300 respectively. The monomer of this protein contains one iron atom per molecule. Rubredoxin has an isoelectric point at pH 2.8 and shows maxima at 570 nm, 490 nm and 370 nm in the oxidized form.During anaerobic sulfide oxidation of a growing culture of Pelodictyon luteolum elemental sulfur is the first main product, which appears in the medium. Elemental sulfur is further oxidized to sulfate, after the available sulfide is completely consumed by the cells.Non-common abbreviations C Chlorobium - P Pelodictyon - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein Offprint requests to: U. Fischer     

A new isolate of Hydrogenobacter,an obligately chemolithoautotrophic,thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 m. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl⁷-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C_18:0, C_18:1 and C_20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Pyruvate — a novel substrate for growth and methane formation in Methanosarcina barkeri

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed.

1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO₂ and CH₄ were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH₄ and CO₂ according to following equation: Pyruvate^-+H⁺+0.5 H₂O » 1.25 CH₄+1.75 CO₂. The molar growth yield (Ych ₄) was about 14 g dry weight cells/mol CH₄. In contrast the growth yield (Ych ₄) of M. barkeri during growth on acctate (Acetate^-+H⁺ » CH₄+CO₂) was about 3 g/mol CH₄.
2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH₄, CO₂ and H₂ (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂. At higher cell concentration less H₂ and CO₂ and more CH₄ were formed due to CH₄ formation from H₂/CO₂. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂ at similar rates and with similar affinity for pyruvate as pyruvate-grown cells.
3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F₄₂₀ was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Thioacetamide as a source of hydrogen sulfide for colony growth of purple sulfur bacteria

Thioacetamide (TAA), CH₃CSNH₂, is an unstable sulfur compound which upon addition of acid decomposes into acetic acid, ammonia, and hydrogen sulfide (H₂S). This characteristic may be taken advantage of when doing plate counts or isolation streaks of phototrophic purple sulfur bacteria. We have performed plate counts and isolation streaks of these bacteria in Gas-Pak anaerobic jars (BBL) supplemented with a test tube containing 0.05 g or 0.10 g TAA dissolved in 1.0 ml of 0.2 N or 0.5 N HCl. It may be demonstrated in a Warburg respirometer that the gas is released over a period of at least 1 week. Colony growth may be observed in 5–10 days. One advantage of this technique is that sodium sulfide (Na₂S·9H₂O) need not be added to the agar medium, which, therefore, may be prepared and stored for future use. This technique has been used successfully for the isolation ofAmoebobacter, Chromatium, Ectothiorhodospira, Lamprocystis, Thiocapsa, andThiocystis.     

Assimilatory sulfur metabolism in Rhodopseudomonas sulfoviridis

Rhodopseudomonas sulfoviridis is unable to grow with sulfate as sole sulfur source. Radioactively labelled sulfate is not incorporated into the cells. Growth only occurs in the presence of reduced sulfur compounds, such as sulfide, thiosulfate, elemental sulfur and cysteine. ATP sulfurylase, adenylylsulfate kinase, O-acetylserine sulfhydrylase and cysteine desulfhydrase are present. Adenylylsulfate sulfotransferase and thiosulfonate reductase are lacking. The enzymes of the sulfate-activating system are not derepressed by O-acetylserine.Non common Abbreviations APS Adenosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate     

Buoyant density changes due to intracellular content of sulfur in Chromatium warmingii and Chromatium vinosum

Average specific density of individual cells of pure cultures of Chromatium warmingii and Chromatium vinosum were measured by isopicnic gradient centrifugation with Percoll during growth at constant illumination as a function of the increasing content of intracellular sulfur. Cell number and volume, bacteriochlorophyll a, sulfide, and sulfur were followed in the cultures along with cellular buoyant density. Poly--hydroxybutyrate was monitored at several points during growth of the cultures. The density of C. warmingii changed from 1.071 to 1.108 g cm^-3 (sulfur content per cell varied from 0 to 1.71pg). C. vinosum changed its density from 1.096 to 1.160 g cm^-3 (sulfur content per cell varied from 0 to 0.43 pg). Maximum sulfur content in pg of sulfur per m³ of cell volume were 0.178 for C. warmingii and 0.294 for C. vinosum. Measurement of the differences in buoyant density, volume and sulfur content before and after ethanol extraction of cells with and without intracellular sulfur, allowed tentatively to estimate the density of sulfur inside the cells as 1.219 g cm^-3. Isolation of sulfur globules and centrifugation in density gradients gave a density higher than 1.143 g cm^-3 for these intracellular inclusions.Non-common abbreviations Bchl Bacteriochlorophyll - DMB Density Marker Beads - PHB poly--hydroxybutyrate     

Sulfur metabolism in Thiorhodaceae. IV. Assimilatory reduction of sulfate byThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711, andChromatium strains 1611 and 6412 can grow with molecular hydrogen replacing sulfide as the electron donor. Sulfate suffices as the sulfur source. The incorporation of radioactive sulfur from³⁵S-sulfate was measured in growing cells in which molecular hydrogen or acetate was the electron donor. In cells pre-grown in sulfide, the incorporation of radioactivity began slowly after a lag period; in contrast, cells grown in sulfate took up the marker at a faster rate and without a lag. The radioactivity appeared in protein as cysteine and methionine. No elimination of sulfide was detected during growth. Thus, the reduction of sulfate was purely assimilatory.