Don P. Kelly

Organic sulfur compounds in the environment : biogeochemistry, microbiology, and ecological aspects

More than a decade has elapsed since the review in Advances by Bremner and Steele (1978) of the role of microorganisms in the atmospheric sulfur cycle. In the intervening decade or so, the dawning realization in the 1970s that volatile organic sulfur compounds are major components of the global sulfur cycle has developed from informed speculation to the status of established fact, supported by ever-accumulating data.

Microbial metabolism of methanesulfonic acid

Abstract 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 ¶Hyphomicrobium 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.

Isolation and physiological characterization of autotrophic sulphur bacteria oxidizing dimethyl disulphide as sole source of energy

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.

Oxidation of Carbon Disulphide as the Sole Source of Energy for the Autotrophic Growth of Thiobacillus thioparus Strain TK-m

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.

Mechanism of Oxidation of Dimethyl Disulphide by Thiobacillus thioparus Strain E6

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.

Chemolithotrophic metabolism of the newly-isolated moderately thermophilic, obligately autotrophic Thiobacillus tepidarius

Thiobacillus tepidarius, isolated from the hot springs at Bath, Avon, UK, grew optimally at 43–45°C and pH 6.0–7.5 on thiosulphate or tetrathionate. In batch culture, thiosulphate was oxidized stoichiometrically to tetrathionate, with a rise in pH. The tetrathionate was then oxidized to sulphate, supporting growth and producing a fall in pH to a minimum of ph 4.8. The organism contained high levels of thiosulphate-oxidizing enzyme, rhodanese and ribulose bisphosphate carboxylase. It was obligately chemolithotrophic and autotrophic. In chemostat culture, T. tepidarius grew autotrophically with the following sole energy-substrates: sulphide, thiosulphate, trithionate, tetrathionate, hexathionate or heptathionate. Thiocyanate, dithionate and sulphite were not used as sole substrates, although sulphite enhanced growth yields in the presence of thiosulphate. Maximum specific growth rate on tetrathionate was 0.44 h-1. ‘True growth yields’ (Ymax) and maintenance coefficients (m) were calculated for sulphide, thiosulphate, trithionate and tetrathionate and observed yields at a single fixed dilution rate compared with those on hexathionate and heptathionate. Mean values for Ymax, determined from measurements of absorbance, dry wt, total organic carbon and cell protein, were similar for sulphide, thiosulphate and trithionate (10.9 g dry wt/mol substrate) as expected from their equivalent oxygen consumption for oxidation. Ymax for tetrathionate (20.5) and the relative Yo values (as g dry wt/g atom oxygen consumed) for thiosulphate and all four polythionates indicated that substrate level phosphorylation did not contribute significantly to energy conservation. These Ymax values were 40–70% higher than any of those previously reported for obligately aerobic thiobacilli. Mean values for m were 6.7 mmol substrate oxidized/g dry wt·h for sulphide, thiosulphate and trithionate, and 2.6 for tetrathionate.

Properties of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus

Abstract 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.

Methanesulphonate utilization by a novel methylotrophic bacterium involves an unusual monooxygenase

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.

Kinetic and Energetic Aspects of Inorganic Sulphur Compound Oxidation by Thiobacillus tepidarius

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.

Physiological Characteristics of a New Thermophilic Obligately Chemolithotrophic Thiobacillus Species, Thiobacillus tepidarius

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.