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Evidence is presented for the presence in propanesulfonate-grown Comamonas acidovorans strain P53 of a cytoplasmically located sulfonatase that does not sediment at 100,000 x g. This enzyme catalysed the sulfonate-dependent oxidation of NADH or NADPH, indicating a monooxygenase that effects the addition of molecular oxygen to C(3)-C(6) 1-alkanesulfonates. Enzyme activity was proportional to protein concentration only above approximately 2 mg cytoplasmic fraction protein ml(-1), suggesting that the sulfonatase is a multicomponent enzyme, possibly comparable with methanesulfonate monooxygenase. Enzyme activity was strongly inhibited by divalent metal-chelating agents, but was insensitive to cyanide and azide. Sulfite released from sulfonates by Comamonas acidovorans was oxidized by an unusual sulfite dehydrogenase. This was purified approximately 230-fold and was shown to have a molecular mass of 74.4 kDa, comprising two or more subunits. The enzyme activity was specific in vitro for ferricyanide as an electron acceptor and, unlike other bacterial sulfite dehydrogenases, did not contain native cytochrome c or reduce added cytochrome c. It was a basic protein, insensitive to chloride and sulfate, and exhibited a K(m) for sulfite of approximately 45mgr;M.

Methanesulfonic acid is a very stable strong acid and a key intermediate in the biogeochemical cycling of sulfur. It is formed in megatonne quantities in the atmosphere from the chemical oxidation of atmospheric dimethyl sulfide (most of which is of biogenic origin) and deposited on the Earth in rain and snow, and by dry deposition. Methanesulfonate is used by diverse aerobic bacteria as a source of sulfur for growth, but is not known to be used by anaerobes either as a sulfur source, a fermentation substrate, an electron acceptor, or as a methanogenic substrate. Some specialized methylotrophs (including Methylosulfonomonas, Marinosulfonomonas, and strains of paragraph signHyphomicrobium and Methylobacterium) can use it as a carbon and energy substrate to support growth. Methanesulfonate oxidation is initiated by cleavage catalysed by methanesulfonate monooxygenase, the properties and molecular biology of which are discussed.

Members of the marigold genus of flowering plants (the genus Tagetes), which synthesize and accumulate thiophene compounds in their roots, were investigated as potential sources of bacteria able to degrade substituted thiophenes. Batch and continuous enrichment cultures inoculated with compost from root balls of Tagetes patula and Tagetes erecta reproducibly produced the same predominant type of bacterium when they were supplied with thiophene-2-carboxylate (T2C) or thiophene-2-acetate (T2A) as a carbon and energy substrate. This organism was a yellow-pigmented, neutrophilic, mesophilic, gram-negative, pleomorphic, rodshaped bacterium, which we classify as a new species of the genus Xanthobacter, Xanthobacter tagetidis; strain TagT2C (= DSM 11105) is the type strain. Strain TagT2CT (T = type strain) grew on simple thiophenes, such as T2C, thiophene-3-carboxylate, and T2A, on analogs of these compounds (pyrrole-2-carboxylate and furan-2-carboxylate), and on the condensed thiophene dibenzothiophene. X. tagetidis was facultatively autotrophic, fixing carbon dioxide by means of ribulose bisphosphate carboxylase, and was able to grow on hydrogen, thiosulfate, or sulfide as an energy substrate. It also grew on a wide range of other heterotrophic, chemolithotrophic, and methylotrophic substrates. Its growth on T2C was optimal at 28 to 31 degrees C and pH 7.6 to 7.8, and the maximum growth rate in batch culture was 0.22 h-1. The DNA base composition of X.tagetidis is 68 mol% G + C. A 16S ribosomal DNA sequence analysis of strain TagT2CT showed that this organism represents a distinct lineage within the Aquabacter-Azorhizobium-Xanthobacter cluster of the alpha-2 subclass of the Pro-teobacteria. Discrimination of X. tagetidis from the other genera in this group and from other Xanthobacter species is discussed.

Summary: A novel methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was the subject of these investigations. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Partial purification of MSAMO from cell-free extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by the mixing of three distinct protein fractions designated A, B and C. The reconstituted enzyme had a narrow substrate specificity relative to crude cell-free extracts. Addition of FAD and ferrous ions to the reconstituted enzyme complex resulted in a fivefold increase in enzyme activity, suggesting the loss of FAD and ferrous ion from the multicomponent enzyme on purification. Analysis of mutants of strain M2 defective in the metabolism of C1 compounds indicated that methanol was not an intermediate in the degradative pathway of MSA and also confirmed the involvement of a multicomponent enzyme in the degradation of MSA by methylotroph strain M2.

Methylotroph strain M2, isolated from soil, was capable of growth on methanesulphonic acid (MSA) as sole carbon and energy source. MSA was oxidized by cell suspensions with an MSA: oxygen stoichiometry of 1.0:2.0, indicating complete conversion to carbon dioxide and sulphate. The presence of formaldehyde and formate dehydrogenases and hydroxypyruvate reductase in MSA-grown bacteria indicated the production of formaldehyde from MSA (and its further oxidation for energy generation), and assimilation of formaldehyde by means of the serine pathway. Growth yields in MSA-limited chemostat culture were a function of dilution rate, with yield ranging from 7.0 g mol-1 at D = 0.04 h-1, to 14.6 at 0.09 h-1. MSA metabolism was not initiated by hydrolysis to produce either methane or methanol, but appears to be by an NADH-dependent methanesulphonate monooxygenase, cleaving MSA into formaldehyde and sulphite. The organism lacked ribulose bisphosphate carboxylase and did not fix carbon dioxide autotrophically. It also lacked ribulose-monophosphate-dependent hexulose phosphate synthase. Growth on methanol, methylammonium and other C1 compounds was exhibited, but ability to oxidize MSA was not induced by growth on these substrates. Similarly, methylammonium (MMA) was only oxidized by strain M2 grown on MMA. Growth on methanol involved a pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase (large subunit molecular mass 60 kDa). This organism is the first methylotroph shown to have the ability to oxidize MSA, by virtue of a novel monooxygenase, and is significant in the global sulphur cycle as MSA can be a major product of the oxidation in the atmosphere of dimethyl sulphide, the principal biogeochemical sulphur gas.

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (Em, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with Em (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The Em (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the ‘445 nm pigment’, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c− → b8 → b182 → c270 → c337 → b322/c360 → O2

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.

Summary: Some kinetic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase from Thiobacillus thyasiris, a marine, facultatively heterotrophic, sulphur-oxidizing bacterium and putative symbiont of Thyasira flexuosa (Montagu), a bivalve mussel, have been determined. The kinetic parameters for the CO2/Mg2+-activated enzyme were: K m(RuBP) 24·3 μM, K m(CO2) 125·5 μM, K m(O2) 900 μM and K m (Mg2+) 1·53 mM. The low CO2 affinity suggests that T. thyasiris may possess a CO2-concentrating mechanism. RuBP oxygenase activity was inhbited by increasing CO2 concentration. Divalent metal ions were essential for RuBP carboxylase activity; activity of the Mg2+-free enzyme could be restored by the addition of Mg2+, Mn2+ or Ca2+. The pH optimum was 7·8. The temperature optimum for RuBP carboxylase activity was 55 °C, although the enzyme rapidly lost activity at this temperature. An Arrhenius plot was biphasic, with a break at 40 °C. The activation energies were 55·5 × 103 J mol-1 and 32·9 × 103 J mol-1> over the temperature ranges 10-40 °C, and 1·47 between 40-55 °C, RuBP carboxylase activity was stable at 35 °C, the optimum growth temperature of T. thyasiris and at 7·5 °C, the temperature of the habitat of Thyasira flexuosa, but the activity was 40% and 3·5%, respectively, of the potential activity at 55 °C. RuBP carboxylase activity was stimulated by NaCl concentrations of up to 0·3 m, with a maximum (33%), occurring between 0·1 and 0·2 m-NaCl. At higher concentrations of NaCl (>0·3 m) RuBP carboxylase activity was inhibited.

Samples of stromatolites, microbial mats, and sediments from four saline lakes (approximate seasonal salinity ranges 20–220%o) in Western Australia were used to establish enrichments for elective cultures of aerobic and anaerobic denitrifying chemolithoautotrophs that could grow with thiosulfate as sole energy source. Organisms of these types were obtained from all sources tested. Twenty‐four pure cultures were isolated, all of which were gram‐negative, rod‐shaped bacteria exhibiting a considerable diversity of metabolic capability. Isolation of these obligate and facultative sulfur‐oxidizing chemolithotrophs from the stromatolite and mat habitats indicates the possibility that these rod‐shaped bacteria contribute to the oxidative phase of the sulfur cycle in these habitats, in addition to oxidation by phototrophs or Beggiatoa. Only four of the pure cultures could grow without salt, but all 24 showed significant halophily, some tolerating 3 M NaCl. Three novel isolates of NaCl‐dependent, thiosulf...

The processes generating and transforming those C1-organosulphur compounds which are major intermediates in the biogeochemical cycling of sulphur are summarised. The biological and chemical interconversions of the methylated sulphides, methane sulphonate, carbon disulphide and carbonyl sulphide are significantly influenced by diverse microorganisms, including autotrophic thiobacilli, methylotrophs, methanogens and sulphate-reducing bacteria. The major biogenic sulphur gas is dimethyl sulphide. A major atmospheric photochemical oxidation product from this is methane sulphonate, which can contribute to the acidity of rain. New data and ideas on the microbiological fate of methane sulphonate and of natural and anthropogenic carbon disulphide are presented.

The halotolerant, facultatively chemolithoautotrophic, gramnegative sulphur-oxidizing organism isolated from the gills of Thyasira flexuosa is shown to exhibit pleomorphism consistent with its being a member of the genus Thiomicrospira and not a Thiobacillus as originally classified. Its distinctive morphology, DNA base composition, and similarity of 16S rRNA sequences to that of Thiomicrospira sp. strain L-12 (which differs only in detail from the type species Thiomicrospira pelophila), lead us to reclassify this strain as Thiomicrospira thyasirae DSM5322.

Thiobacillus halophilus and Thiobacillus hydrothermalis share 98.7% similarity in 16S rRNA sequence, possess similar gross DNA composition (64.2 and 67.4 mol% G+C values, respectively), and have similar physiological properties. While this might have indicated that they were strains of a single species, DNA-DNA hybridization between the type strains of the two species showed only 59% hybridization, indicating the organisms to be different at the species level. Thiobacillus neapolitanus is the phylogenetically nearest neighbour of T. halophilus and T. hydrothermalis (91.6-92.1% similarity in 16S rRNA sequence) and is the only other Thiobacillus in the gamma-subclass of the Proteobacteria that can be regarded as exclusively related to these two species. The 16S rRNA gene sequences of these three species are so different from those of the other thiobacilli in the gamma-subclass that they justify recognition as a distinct phyletic group. Their comparative properties are summarized.

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase [3 μmol NADH oxidized min–1 (mg crude extract protein)–1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [V max 634 ± 24 nmol NH3 formed min–1 (mg protein)–1] than on cyanate [89 ± 9 nmol NH3 min–1 (mg protein)–1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (K m 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (K i 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

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 (Ymax) 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 (μmax) 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.

Total base sequences of the 16S rRNA genes of Thiobacillus halophilus and Thiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative of T. halophilus is Thiobacillus hydrothermalis (with 98.7% similarity), and the closest relative of T. aquaesulis is Thiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genus Thiobacillus are clearly so diverse that they need reclassification into several genera. The type species, T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus name Thiobacillus be restricted to the chemolithoautotrophic species falling into that group. T. aquaesulis and T. thioparus may thus be regarded as true species of Thiobacillus. The relatively large number of obligately chemolithoautotrophic Thiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.

Sulfonates. These are organosulfur compounds in which sulfur at the oxidation state of +5 is covalently linked to a carbon atom, which may be part of a larger carbon based molecule: R-CH2-SO3H. The simplest sulfonate is methanesulfonic acid, where R = H, and it and the series of aliphatic sulfonates, CH3.(CH2) n .SO3H, in which n = 1-12, have been shown to be used as sources of sulfur for growth by various microorganisms (Thysse, Wanders 1972; Biedlingmeier, Schmidt 1982; Krauss, Schmidt 1987). More complex sulfonates include the naturally occurring cysteate, taurine, isethionate, and N-acylaminosulfonates and anthropogenic aromatic sulfonates, such as substituted naphthalenesulfonic and benzenesulfonic acids, and pH buffers such as HEPES (N-[2-hydroxyethyl]-piperazine-N’-[2-ethanesulfonate]). Some of these can be degraded by bacteria for use as sources of sulfur, and in some cases also as sources of both carbon and energy for growth (Locher et al. 1989; Seitz et al. 1993; Kertesz et al. 1994; Chien et al. 1995).

Publisher Summary This chapter describes various techniques that have been used in studies of sulfur compound oxidation and energy coupling using suspensions of intact organisms and for the separate identification and characterization of enzymes that are located in the periplasm of thiobacilli . Separation of the periplasmic fractions from both Thiobacillus versutus and Thiobacillus tepidarius has been achieved by methods based on a procedure developed for Escherichia coli . Techniques developed for the assessment of amidination of the inner and outer membrane surfaces of human erythrocytes can be adapted for use with gram-negative bacteria to determine whether proteins are located in the periplasm or inside the cytoplasmic membrane of the cell. Monitoring of the time course of the oxidation of thiosulfate labeled in either the sulfane (S–) or sulfonate (–SO 3 ) atom can also be informative of the mechanism of initial attack on thiosulfate and the possible formation of intermediates of the process.

Publisher Summary This chapter discusses synthesis and determination of thiosulfate and polythionates. Thiosulfate and polythionates react with cyanide to form thiocyanate, which can be determined colorimetrically as ferric thiocyanate. Differences in the reactivity of the thionates with cyanide enable the quantitative characterization and determination of mixtures of several compounds: trithionate is stable at high pH values and reacts with cyanide only at elevated temperatures, thiosulfate reacts with cyanide at room temperature only in the presence of copper(II) as a catalyst, whereas the higher polythionates react rapidly with cyanide at room temperature to form thiosulfate and sulfite. Thiocyanate formation can be quantified by reference to a calibration curve using thiocyanate or thiosulfate standards. In all the procedures in which ferric thiocyanate is measured, caution needs to be exercised, as the color is light sensitive and readings should be made at once or samples stored in darkness after addition of ferric reagent. The sensitivity of the procedure can be enhanced by using small volumes of more concentrated reagents and reading optical density in long light path (4 cm) cuvettes.

Publisher Summary This chapter describes assay procedures for a variety of enzymes occurring in lithotrophs and some heterotrophs. Some of these enzymes, such as adenylylsulfate (APS) reductase and thiosulfate reductase also occur in sulfate-reducing bacteria (SRB). Thiosulfate and polythionates serve as energy-yielding or electron-donating substrates in the metabolism of a wide range of chemolithotrophic and photolithotrophic bacteria, as well as some heterotrophs. Thiosulfate dehydrogenase is purified from a number of different Thiobacillus spp. and found to vary greatly in structural and catalytic properties. The activity of trithionate hydrolase was first demonstrated in Thiobacillus neapolitanus ( T. neapolitanus ) and the enzyme subsequently was recovered from both Thiobacillus tepidarius ( T. tepidarius ) and Thiobacillus acidophilus ( T. acidophilus ). The enzyme can be determined in a coupled assay with thiosulfate dehydrogenase, using 0.1 m M sodium trithionate instead of thiosulfate, and measuring thiosulfate-dependent cytochrome c reduction at pH 7.0. Thiosulfate produced by the hydrolase becomes the substrate for an excess of thiosulfate dehydrogenase and the rate of reduction of cytochrome c equates to the specific activity of the hydrolase.

Four eubacterial strains able to grow on carbon disulfide (CS2) as sole energy substrate were isolated from soil and leaves of the CS2-producing tree Quercus lobata. Three of the isolates (strains KS1, KS2, and KL1) were gram-negative, facultatively methylotrophic, and heterotrophic, and capable of growth on a wide range of inorganic and organic sulfur compounds. Biochemical and physiological properties differed slightly among the three strains, but all are proposed to be novel thiobacillus species. Growth yields on CS2 in batch and chemostat culture ranged from 3.3 g dry wt/mol CS2 (batch) to a maximum growth yield (Ymax) of 11.1 g dry wt/mol (chemostat). Chemostat data for two of the strains growing, autotrophically on thiosulfate gave Ymax values of 7.4 and 7.1 g dry wt/mol, which fall within the range observed with thiobacilli. The three new Thiobacillus strains had DNA containing 39.8 (KS2), 47.8 (KS1), and 50.5 (KL1) mol% G+C. All three were unusual in being able to grow not only on thiosulfate (aerobically or with denitrification), but also on CS2, carbonyl sulfide and methylated sulfides as sole energy substrates, and one was unique in being able to grow also on substituted thiophenes. They are the first organisms described to be capable, of anaerobic growth with denitrification on CS2. The fourth isolate (strain KL2) was gram-positive non-motile and nonspore-forming, with 39.0 mol% G+C. It had a restricted range of sulfur-containing growth substrates, could not grow methylotrophically or on autotrophic substrates other than CS2, and is not yet classifiable These organisms extend the range of eubacteria known to be capable of CS2 breakdown and demonstrate that several types of facultatively chemolithotrophic bacteria, able to grow exclusively on CS2, are associated with a CS2-producing plant.

Summary: The isolation of a number of strains of bacteria able to grow on dimethyl disulphide and dimethyl sulphide as sole source of energy is described. The isolates came from diverse habitats, including soil, peat, marine mud and a freshwater pond. The isolates were morphologically and physiologically best described as thiobacilli, capable of growth as Calvin cycle autotrophs on inorganic sulphur compounds, methylated sulphides or thiocyanate. They could not grow heterotrophically or methylotrophically. One isolate (E6) was examined in detail. Substrate oxidation kinetics indicated that methanethiol, sulphide, formaldehyde and formate, but not dimethyl sulphide, could be implicated as intermediates in dimethyl disulphide metabolism. Apparent K s values for the oxidation of dimethyl disulphide and methanethiol were 2.5 and 3.2 μM respectively. Growth yields in chemostat culture on dimethyl disulphide with and without thiosulphate indicated that energy conservation was probably coupled to the oxidation of formaldehyde and sulphide (derived from dimethyl disulphide via methanethiol) to CO2 and sulphate. Maximum growth yield (Y max) on dimethyl disulphide was 17 g cell-carbon per mol of dimethyl disulphide. At one dilution rate (0.078 h−1), the biomass of a culture limited by dimethyl disulphide increased when thiosulphate was also supplied, indicating a thiosulphate-dependent yield of 2.45 g cell-carbon mol−1. This is the first demonstration of the isolation of organisms into pure culture that are capable of growth on dimethyl disulphide as sole energy substrate, and of degrading it completely to CO2 and sulphate.

Dimethyl sulphide (DMS) was degraded by acclimatized activated sludge and by a mixed culture of Thiobacillus thioparus TK-1 and Pseudomonas sp. AK-2. While both these organisms persisted in stable co-culture on DMS, it was found that T. thioparus TK-1 and the derived strain TK-m grew in pure culture on DMS, and oxidized DMS with an apparent Km of 4.5 × 10−5 M. During growth, all the DMS-sulphur was oxidized stoichiometrically to sulphate but no methanol was detected in pure cultures of TK-m. DMS-carbon was probably converted to CO2, since the fixation of 14CO2 was progressively diluted during growth of a culture on 14CO2 and DMS. Growth yields were consistent with autotrophic growth, dependent on the oxidation of the methyl residues to CO2 (probably with formaldehyde as a first intermediate) and the sulphide to sulphate. The organism thus appears to exhibit a mixture, from the one substrate, of chemolithotrophic and methylotrophic energy generation supporting autotrophic growth with CO2 fixation.

In aerobic chemostat culture Thiobacillus A2-GFI grew autotrophically on formate and heterotrophically on glucose with maximum specific growth rates (μmax) of 021 and 033 h-1, respectively. At dilution rates of 01 and 018 h-1, it grew mixotrophically on formate + glucose mixtures, completely consuming both substrates. Ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase were present at high specific activity in autotrophic and mixotrophic cultures, but were repressed in cultures on glucose alone. A greater proportion of added glucose was assimilated in mixotrophic culture than in heterotrophic culture. Raising the dilution rate of a mixotrophic culture from 01 or 018 to 03 h-1 resulted in washout (with an apparent μmax for mixotrophic growth of 025 h-1) and the establishment of a culture dependent on glucose for growth. Growth yields on formate and glucose were, respectively, 33 and 100 g dry wt (mol substrate consumed)-1. Steady state biomass production in mixotrophic culture indicated additive growth yields. The biomass produced in cultures on formate + glucose at a dilution rate of 03 h-1 suggested that growth only occurred on glucose, but organisms still contained high activities of ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase. At a formate: glucose ratio (mm) of 100:1, some formate was oxidized and CO2 was fixed, but formate was not used when this ratio was 50:5. Formate-glucose mixotrophy benefits Thiobacillus A2-GFI when substrates are limited at low growth rates (< μmax for formate), but is characterized by a μmax below that possible on glucose. Physiological behaviour at high growth rates was influenced by the formate:glucose ratio, resulting under some conditions, at least, in loss of mixotrophy and the establishment of heterotrophic growth.

Bacteria have been isolated into pure culture from the gills ofLucinoma borealis, Myrtea spinifera, Thyasira flexuosaandT. sarsicollected from several British and Scandinavian coastal sediments. It is proposed that these bacteria are symbiotic with the animals. The bacteria were either autotrophs, obtaining energy from oxidation of inorganic sulphur compounds, or methylotrophs using methanol or methylamine as the source of carbon and energy. The latter could also oxidize thiosulphate to tetrathionate and precipitate elemental sulphur. The pure cultures have been characterised in some detail, showing that (1) their DNA base composition falls in the range 47–58% guanine + cytosine; (2) although all grow well at the natural environment temperatures of 8–15°C, their optima lie between 30 and 35°C; (3) all produce copious amounts of extracellular slime when grown at low temperatures; (4) the autotrophic isolate is facultatively heterotrophic, containing a repressible ribulose bisphosphate carboxylase, and excreting up to 30% of its carbon dioxide fixation into the culture medium; (5) the methylotrophs include some containing high activities of hexulose phosphate synthase for assimilation of formaldehyde by the ribulose monophosphate cycle; others may have novel assimilatory metabolism. The possibility is discussed that environmental factors may determine whether a methylotroph-animal or autotroph-animal symbiosis is established rather than the symbiont type being an invariable species-specific character.

Comparison of the effects of uncoupling agents, arsenate, fluoride and cyanide on sugar transport byThiobacillus A2 indicated that (a) glucose uptake by fast-growing strain GFI involved a high energy phosphate dependent system; (b) transport of glucose by the wild type and of fructose and sucrose by both strains, was by respiration dependent systems requiring a membrane proton gradient. Glucose and sucrose were competitive inhibitors of the transport of each other. Bacteria grown on glucose +sucrose (20+2 mM) transported glucose much faster than those grown on glucose alone. This could explain the great stimulation of growth rate on glucose, in the presence of sucrose.

Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiobacillus versutus in chemostat culture. In the thiosulphate-oxidizing enzyme system of T. versutus, protein B was the only component found to contain manganese in significant amounts. When it was examined by electron spin resonance (ESR) spectroscopy, protein B gave a broad, complex spectrum, indicative of the presence of a dimeric manganese cluster, with manganese in the Mn(II) oxidation state.

SUMMARY: The enzyme oxidizing thiosulphate to tetrathionate (‘tetrathionate synthase’) has been purified: the native enzyme has a M r of 138000 and subunit M r of 45000, contains no haem and shows K m values for thiosulphate of 4 and 110 μM respectively with cytochrome c or ferricyanide as electron acceptor. Most of the enzyme was recovered from the periplasm of the cell. Evidence is also presented for a trithionate hydrolyase, catalysing the hydrolytic cleavage of trithionate to thiosulphate and sulphate. This activity was partially purified and assayed in a coupled system in which cytochrome c reduction by the purified tetrathionate synthase was measured. Tetrathionate oxidation by cell-free preparations could not be obtained, but a sulphite dehydrogenase (with ferricyanide as electron acceptor) was demonstrated and sulphite-dependent reduction of cytochrome c in T. tepidarius membrane preparations occurred. The latter was inhibited by HQNO (2-heptyl-4-hydroxyquinoline-N-oxide), indicating the involvement of cytochrome b. Sulphite oxidation by APS reductase (adenylylsulphate reductase) could not be detected, which was consistent with the earlier demonstration of complete inhibition of ATP synthesis in intact organisms by FCCP (carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone). A scheme is presented which is consistent with all the whole organism and enzyme data available to date.

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and alpha-linked disaccharides, but not on beta-linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occured on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.

Summary: The ability of micro-organisms to grow on carbon disulphide (CS2) as a sole source of carbon and energy appears to be very limited: none was obtained from enrichment culture and eight Thiobacillus species could not use it. Thiobacillus thioparus strain TK-m could grow autotrophically on either CS2 or carbonyl sulphide (COS) as sole substrates. Growth yield on CS2 was 7.9 ± 0.9 g cell-carbon (mol CS2)−1, and yields on COS, thiosulphate or thiocyanate were in the range 5.6—6.1. COS was detected as an intermediate during growth on CS2, and there was quantitative conversion of the sulphur of CS2 to sulphate during growth. Aerobic oxidation of CS2 by suspensions of strain TK-m exhibited a K s of 16.5 μM and a V max of 524 nmol O2 consumed min−1 (mg organism-protein)−1. When incubated anaerobically with CS2, strain TK-m sequentially produced COS and H2S. CS2 oxidation is proposed to proceed by its sequential hydrolytic cleavage to COS then H2S, with release of all the carbon as CO2, followed by oxidation of the sulphide to sulphate. This oxidation provides all the energy for growth, which is dependent on the autotrophic fixation of CO2, apparently by means of ribulose bisphosphate carboxylase.

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.

Enzyme A of the thiosulphate-oxidizing multi-enzyme system of Thiobacillus versutus was shown by dialysis equilibrium, using [35S]thiosulphate, to bind 1 mole of thiosulphate per mole of enzyme. The formation of this complex is believed to the the first step of the thiosulphate-oxidizing sequence, leading to sulphate. Sulphite was a competitive inhibitor of thiosulphate oxidation by the multi-enzyme system, with an apparent K1 of 25 μM, and was also an inhibitor of thiosulphate binding to enzyme A, with an apparent Kd of 70 μM. Activity of the multi-enzyme system was inhibited by sulphydryl group reagents, indicating the importance of SH groups in the overall oxidation process. Inhibition by N-ethylmaleimide, p-hydroxymercuribenzoate and HgCl2, but not by iodoacetamide, was reversed by reduced glutathione. SH group reagents did not inhibit the binding of thiosulphate to enzyme A, supporting the indication from sulphite-inhibition of binding that binding was effected through the sulphonate group of thiosulphate and did not directly involve free sulphydryl groups. Dithionate and methane sulphonate did not affect binding by enzyme A or thiosulphate oxidation by the multi-enzyme system. The overall stoichiometry of thiosulphate oxidation resulted in 8 mol cytochrome c being reduced for each thiosulphate ion oxidized, thereby eliminating the possibility that an oxygenase was involved in the system. A hypothetical scheme describing thiosulphate binding and oxidation is presented.

We describe a new Thiobacillus species which is a gram-negative, motile, rod-shaped organism having polar flagella. The optimum growth temperature is 43 to 45°C, and the optimum pH range is 6.0 to 7.5. This organism is obligately chemolithotrophic and autotrophic and has ribulose bisphosphate carboxylase activity. It is able to oxidize thiosulfate, trithionate, tetrathionate, hexathionate, heptathionate, sulfur, and sulfide, but is not able to use sulfite, thiocyanate, or dithionate for growth. In batch culture it converts thiosulfate to tetrathionate during or before growth. It has both rhodanese and thiosulfate-oxidizing enzyme activites. It does not grow anaerobically with nitrate or nitrous oxide on either thiosulfate or tetrathionate. The guanine-plus-cytosine content of its deoxyribonucleic acid is 66.6 mol%, and it contains ubiquinone Q-8 in its respiratory chain. The organism is named Thiobacillus tepidarius sp. nov. The type strain is strain DSM 3134, which has been deposited in the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany.

ApparentKs andVmax values, for the transport byThiobacillus A2 of14C-labelled sucrose, hexoses and pentoses, were estimated using flow dialysis and membrane filtration techniques. Transport systems of varying degrees of specificity could be inferred from the data. For most sugars tested including glucose, fructose and arabinose, there was a close correlation between maximum rate of sugar transport and observed growth rate. Differences in transport rate were sufficient to explain slow and fast growth on glucose by wild type and GF strains ofThiobacillus A2.

Thiobacillus A2 was grown in chemostat culture under four distinct types of substrate limitation: chemolithoautotrophically with limitation by thiosulphate or CO2; heterotrophically with limitation by glucose; and mixotrophically with dual limitation by both thiosulphate and glucose. Under mixotrophic conditions energy was obtained from the oxidation of both thiosulphate and glucose, and carbon was derived both from CO2 fixation by the Calvin cycle and from glucose. Ribulosebisphosphate carboxylase (RuBP carboxylase) activity was negligible and chemolithotrophic thiosulphate oxidation and autotrophic CO2 fixation were apparently repressed in bacteria which had been grown heterotrophically. Conversely, under autotrophic conditions the ability to oxidize glucose was repressed. Growth yields from mixotrophic cultures were the sum of those obtained under single substrate limitation. Intermediate activities of RuBP carboxylase were detected in mixotrophic cultures, but more glucose was assimilated mixotrophically than heterotrophically. Glucose was metabolized by the Entner-Doudoroff (85 to 90%) and pentose phosphate (10 to 15%) pathways under both heterotrophic and mixotrophic conditions, with slight involvement also of the Embden-Meyerhof pathway (

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the →H+/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The →H+/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a →H+/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa3, does not function as a proton pump.

SUMMARY: Several thiobacilli and Thermothrix thiopara were grown in chemostat culture with inorganic sulphur compounds as growth-limiting energy substrates for autotrophic growth. Thiobacillus neapolitanus, Thiobacillus thiooxidans and Thiobacillus acidophilus were all able to use thiosulphate, trithionate or tetrathionate as sole energy substrates, as was Thx. thiopara grown at 72 °C. “True growth yields” (Y max) were estimated for the organisms and showed yields of T. neapolitanus to be the same on trithionate and thiosulphate, thereby suggesting that only oxidative, and not substrate-level, phosphorylation is involved in energy conservation in this organism. The yield data suggested that substrate-level phosphorylation could, however, be significant in T. acidophilus. Thiobacillus versutus only grew with thiosulphate, with which it gave yield values similar to those of T. neapolitanus and T. thiooxidans. Thx. thiopara exhibited maximum specific growth rates on thiosulphate, trithionate and tetrathionate of 0·55, 0·23-0·25 and

A moderately thermophilic, facultatively chemolithoautotrophic thiobacillus isolated from a thermal sulphur spring is described. It differs from all other species currently known to be in culture. It grows lithoautotrophically on thiosulphate, trithionate or tetrathionate, which are oxidized to sulphate. Batch cultures on thiosulphate do not produce tetrathionate, but do precipitate elemental sulphur during growth. In autotrophic chemostat cultures the organism produces yields on thiosulphate, trithionate and tetrathionate that are among the highest observed for a Thiobacillus. Autotrophic cultures contain ribulose bisphosphate carboxylase. Heterotrophic growth has been observed only on complex media such as yeast extract and nutrient broth. It is capable of autotrophic growth and denitrification under anaerobic conditions with thiosulphate and nitrate. It grows between 30 to 55° C, and pH 7 to 9, with best growth at about 43°C and pH 7.6. It contains ubiquinone Q-8, and its DNA contains 65.7 mol% G+C. The organism is formally described and named as Thiobacillus aquaesulis.

Summary: A recently isolated organism, capable of chemolithoautotrophic growth on dimethyl disulphide, was characterized as a strain of Thiobacillus thioparus. It had DNA with a base composition of 60.5 ± 1.0 mol% G + C, and ubiquinone-8 (UQ-8) as its only respiratory quinone. Its growth in chemostat culture (at a growth rate of 0.07-0.08 h−1) showed yields of 14.4, 11.8 and 2.45 g cell-carbon per mol of dimethyl disulphide (DMDS), dimethyl sulphide (DMS) and thiosulphate, respectively. This is consistent with energy generation from the oxidation of the methyl and the sulphide moieties of DMDS, with oxidation of sulphide to sulphate contributing a yield of about 2.8 g cell-carbon mol−1. From whole organism and cell-free extract studies, DMDS oxidation was shown to proceed by its (NADH-stimulated) reduction to methanethiol (MT), which was oxidized via sulphide, formaldehyde and formate to CO2 and sulphate by MT oxidase, formaldehyde and formate dehydrogenases, and an uncharacterized sulphide-oxidizing system. The MT oxidase had a K m of 9.7 μM and showed substrate inhibition with a K i of about 8 μM. The essential role of catalase during growth on DMDS was shown by the sensitivity of growth on DMDS (but not on thiosulphate) to 3-amino-1,2,4-triazole. Catalase is believed to destroy the peroxide produced by the MT oxidase reaction. DMDS-grown organisms oxidized sulphide, thiosulphate and tetrathionate (with the latter indicated to be an intermediate in thiosulphate oxidation), suggesting the pathway of sulphide oxidation to be similar to that in some other thiobacilli. Carbon assimilation was by the Calvin cycle, with ribulose bisphosphate carboxylase being present in cell-free extracts at a specific activity of 80 nmol CO2 fixed min−1 (mg protein)−1. Hydroxypyruvate reductase (HPR) was not detected at levels sufficient to indicate any role in primary carbon assimilation.

Etude physiologique de 3 souches dont l'une n'a pas encore ete decrite, comparaison avec les Thiobacillus

Summary: A detailed radiorespirometric and enzymological analysis was made of the wild-type and GFI strains of Thiobacillus A2 grown on numerous sugars. The wild-type used the Embden-Meyerhof, Entner-Doudoroff and pentose phosphate pathways for glucose oxidation only after culture on glucose. The fast-growing strain GFI used all three pathways after growth on glucose, maltose or fructose. The wild-type grown on a wide range of pentoses, hexoses, disaccharides, a trisaccharide and a mixture of glucose and maltose oxidized glucose by means of the Entner-Doudoroff pathway (68 to 90%) and pentose phosphate pathway (10 to 32%). The key enzyme determining the presence or absence of the Embden-Meyerhof pathway was 6-phosphofructokinase. Other mechanisms, such as a phosphoketolase pathway, were shown to be unimportant, even during growth on pentoses. Arabinose was metabolized via hexose phosphate synthesis. The mechanisms of regulation of sugar metabolism and the energetic significance of alternative pathways are discussed.

SUMMARY: Whole organisms of Thiobacillus tepidarius oxidize thiosulphate to sulphate with the obligatory formation of tetrathionate as an intermediate. Oxidation of thiosulphate to tetrathionate shows an apparent K m of about 120 μM and is relatively insensitive to FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), HQNO (2-heptyl-4-hydroxyquinoline-N-oxide) or thiocyanate. Oxidation of tetrathionate to sulphate shows a K m of about 27 μM and is strongly inhibited by FCCP, HQNO, thiocyanate and gramicidin. Sulphite oxidation is also inhibited by FCCP and HQNO. Trithionate oxidation to sulphate occurred and showed unexplained dependence on the presence of sulphate ions. A H+/O quotient of about 4 for proton translocation driven by substrate oxidation was seen for each of thiosulphate, tetrathionate and sulphite. ATP synthesis coupled to thiosulphate oxidation was completely abolished by FCCP. The results obtained are consistent with the oxidation of thiosulphate (and probably trithionate) to tetrathionate in the periplasm of the cell, with HQNO-insensitive electron transport to cytochrome c, and with further oxidation of tetrathionate (and sulphite) to sulphate after FCCP-sensitive transport to the cytoplasmic side of the membrane. The latter oxidations involve HQNO-sensitive electron transport via cytochrome b. Inhibition of tetrathionate metabolism by thiocyanate and gramicidin would be consistent with tetrathionate transport by a S4O-/4H+ symport process. The proton translocation experiments indicate the mechanism of H+ extrusion to depend on electron transfer within the quinone/cytochromes bc segment of the respiratory chain, and does not involve a proton-pumping oxidase. The sulphur-compound-oxidizing system of T. tepidarius is shown to be quite different from that previously described for T. versutus.

Four c -type cytochromes were purified by several procedures including chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephadex G-75, G-100 and G-200 and chromatofocusing. Cytochrome c -551 had a p I value of 5.2 and an M r of 260 000 consisting of six non-covalently bound polypeptides each with an M r of 43 000, and contained four to five haems. Cytochrome c -552.5 had a p I value of 4.8 and an M r of 56 000 consisting of two polypeptides with the same M r 29 000, and contained two haems. Cytochromes c -551 and c -552.5 were reduced by ascorbate to about 70 and 60% of the fully dithionite-reduced values, respectively, and both were essential components in the thiosulphate-oxidizing multi-enzyme system (other components of the system were ‘enzyme A’, ‘enzyme B’ and sulphite: cytochrome c oxidoreductase). These two cytochromes functioned as electron carriers and effectors in the oxidation of thiosulphate. Some evidence suggested that cytochrome c -551 might be a specialized electron transfer component for sulphonate-sulphur oxidation. Both cytochromes could be reduced by thiosulphate in the presence of enzymes A and B. Cytochrome c -550 (basic) and cytochrome c -550 (acidic) were small proteins with M r 15 000 and 14 000 and p I values of over 8 and 5, respectively. Their physiological role is uncertain.

Thiobacillus versutus responds to both CO2-limitation and increase in chemostat dilution rate under thiosulphate-limitation by increasing ribulose bisphosphate carboxylase specific activity. It has no high affinity CO2-concentrating mechanism like that shown in Synechococcus, and may depend on diffusional uptake of CO2/HCO3−.

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by 14C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.