Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Studies on the metabolism of a sulfur-oxidizing bacterium VI1. Fractionation and reconstitution of the elementary sulfur-oxidizing system of Thiobacillus thiooxidans

The sulfur-oxidizing system of a strain of Thiobacillus thiooxidanswas obtained in cell-free state. The system is resolved intothree fractions and can be reconstituted from these fractions.Both the soluble and particulate fractions are required forthe oxidation of elementary sulfur. The soluble fraction wasfurther separated into two fractions, the collodion membrane-permeable(S-P)and the impermeable(S-IP). S-P contains a low molecular weight,relatively heat stable substance(s) which is indispensable forthe reconstitution of the sulfur-oxidizing system and was identifiedas a pyridine nucleotide. The function of S-P can be replacedby NAD or NADP, but not by cysteine nor GSH. Oxidation of NADH₂ and NADPH₂ is catalyzed by the particulatefraction. Oxidation of the latter is much more rapid than thatof the former. Oxidation of NADPH₂ as well as sulfur oxidationis inhibited by cyanide, pCMB and CO, the CO-inhibition beingphoto-irreversible. However, strong inhibitors of sulfur oxidationsuch as DDC, 8-hydroxyquinoline and salicylaldoxime have noeffect on the oxidation of NADPH₂. The optimum pH values for sulfur and sulfite oxidations by thecell-free extract are shifted to the neutral side in comparisonwith pH values by intact cells. ¹V = References(I). ²Partly supported by a grant from the Ministry of Education. (Received April 3, 1969; )     

Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans

Thiobacillus thiooxidans was grown at pH 5 on thiosulfate as an energy source, and the mechanism of oxidation of inorganic sulfur compounds was studied by the effect of inhibitors, stoichiometries of oxygen consumption and sulfur, sulfite, or tetrathionate accumulation, and cytochrome reduction by substrates. Both intact cells and cell-free extracts were used in the study. The results are consistent with the pathway with sulfur and sulfite as the key intermediates. Thiosulfate was oxidized after cleavage to sulfur and sulfite as intermediates at pH 5, the optimal growth pH on thiosulfate, but after initial condensation to tetrathionate at pH 2.3 where the organism failed to grow. N-Ethylmaleimide (NEM) inhibited sulfur oxidation directly and the oxidation of thiosulfate or tetrathionate indirectly. It did not inhibit the sulfite oxidation by cells, but inhibited any reduction of cell cytochromes by sulfur, thiosulfate, tetrathionate, and sulfite. NEM probably binds sulfhydryl groups, which are possibly essential in supplying electrons to initiate sulfur oxidation. 2-Heptyl-4-hydroxy-quinoline N-oxide (HQNO) inhibited the oxidation of sulfite directly and that of sulfur, thiosulfate, and tetrathionate indirectly. Uncouplers, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), inhibited sulfite oxidation by cells, but not the oxidation by extracts, while HQNO inhibited both. It is proposed that HQNO inhibits the oxidation of sulfite at the cytochrome b site both in cells and extracts, but uncouplers inhibit the oxidation in cells only by collapsing the energized state of cells, delta muH+, required either for electron transfer from cytochrome c to b or for sulfite binding.     

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

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

Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil

Two heterotrophic bacteria that oxidized thiosulfate to tetrathionate were isolated from soil. The enzyme system in one of the isolates (C-3) was constitutive, but in the other isolate (A-50) it was induced by thiosulfate or tetrathionate. The apparent K(m) for oxygen for thiosulfate oxidation by A-50 was about 223 mum, but, for lactate oxidation by A-50 or thiosulfate oxidation by C-3, the apparent K(m) for oxygen was below 2 mm. The oxidation of thiosulfate by A-50 was first order with respect to oxygen from 230 mum. The rate of oxidation was greatest at pH 6.3 to 6.8 and at about 10 mm thiosulfate, and it was strongly inhibited by several metal-binding reagents. Extracts of induced A-50 reduced ferricyanide, endogenous cytochrome c, and mammalian cytochrome c in the presence of thiosulfate. A-50, once induced to oxidize thiosulfate, also reduced tetrathionate to thiosulfate in the presence of an electron donor such as lactate. The optimal pH for this reaction was at 8.5 to 9.5, and the reaction was first order with respect to tetrathionate. There was no correlation between the formation of the thiosulfate-oxidizing enzyme of A-50 and the incorporation of thiosulfate-sulfur into cell sulfur. Thiosulfate did not affect the growth rate or yield of A-50.     

Studies on Biochemistry of the Thiobacilli

In the oxidation of thiosulfate at pH 4.5 tetrathionate was formed as an intermediate, and the thiosulfate-oxidizing enzyme was active in acidic pH range in contrast to the enzyme of T. thioparus and Thiobacillus X.

Phosphate did not seem to affect the oxidation of thiosulfate but rather affect the conversion of tetrathionate. In the absence of phosphate, tetrathionate, which was produced from thiosulfate oxidation, seemed to accumulate without undergoing further conversion.

Quantitative oxidation of tetrathionate to sulfate was achieved with freshly harvested cells of T. thiooxidans; pH optimum for the oxidation of tetrathionate by the washed cells was 2~3, and the activity fell markedly at pH above 3.5.

Tetrathionate might be enzymatically dismuted to pentathionate and trithionate under anaerobic conditions with crude extracts of T. thiooxidans; pH optimum for the reaction was about 2.7 and the activity fell strikingly at pH 4.7. The formed trithionate might be further hydrolyzed to thiosulfate and sulfate.     

Reduced sulfur compound oxidation by Thiobacillus caldus.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.     

Studies on the metabolism of a sulfur-oxidizing bacterium VII. Oxidation of sulfite by a cell-free extract of Thiobacillus thiooxidans

Properties of the cell-free extract, prepared from a strainof Thiobacillus thiooxidans by sonic disruption followed byfractionation with centrifugatiori, were investigated with referenceto its sulfite-oxidizing activity. Without the addition of cofactors the particulate fraction(F-P)catalyzed oxidation of sulfite with oxygen or bacterial cytochromec-552 obtained from Pseudomonas stutzeri as electron acceptor.TMPD reduced by ascorbic acid was also oxidized by F-P. Thesoluble fraction(F-S) showed no activity in oxidizing sulfiteand TMPD, but stimulated TMPD oxidation by F-P. Oxygen uptake with either sulfite or TMPD as substrate was inhibitedby KCN, NaN₃, CO and c-phenanthroline. CO-Inhibition was reversedby light. Reduction of cytochrome c-552 by sulfite was insensitiveto these agents. Antimycin A markedly inhibited sulfite oxidation with eitheroxygen or cytochrome c-552 as electron acceptor, but was withouteffect on TMPD oxidation. DDC and SAO, both strong inhibitors of sulfur oxidation, didnot affect sulfite and TMPD oxidations. Cytochromes of the a, b and c types were contained in F-P. Thesecytochromes were rapidly reduced when F-P was incubated withsulfite. Cytochrome(s) of the c type was present in F-S, too. ¹VI.=References (3) ²Partly supported by a grant from the Ministry of Education ³Present address: Sanyo Women's College, Hatsukaichi, Hiroshima738, Japan ⁴Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima 734, Japan (Received May 15, 1970; )     

Kinetics of sulfur oxidation by thiobacillus ferrooxidans

Abstract

Thiobacillus ferrooxidans ATCC 23270 was grown with elemental sulfur as the energy source. Substrate oxidation was measured using a Clark‐type oxygen electrode. Whole cells demonstrated a broad pH optimum for sulfur oxidation between pH 2.0 and 8.0. The V _max and K_sfor sulfur oxidation varied depending on pH. Sulfite was oxidized at 227 nmol O₂/min/mg protein. Thiosulfate oxidation was slow, and tetrathionate oxidation was not detected. At a concentration of 2 mM, sodium azide completely inhibited sulfur, sulfite, and thiosulfate oxidation. Inhibition by N‐ethylmaleimide, antimycin A, and 2‐heptyl‐4‐hydroxyquinoline N‐oxide varied with substrate.     

The role of thiosulfate in sulfur metabolism of Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to assimilate both sulfur moieties of thiosulfate. During growth on ³⁵S-labelled thiosulfate the amino acids cysteine, homocysteine and methionine were labelled. The bulk of thiosulfate, however, was oxidized to tetrathionate and accumulated in the medium. A thiosulfate: acceptor oxidoreductase was partially purified and characterized. The enzyme oxidized thiosulfate to tetrathionate in the presence of ferricyanide. A c-type cytochrome isolated from this organism was reduced by this enzyme.     

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.     

Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate

Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autotrophic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studies, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidation by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO₂-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities of enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.     

Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate.

Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum oxidation rates at pH 3.0, but produced substrate inhibition at other pH values. Tetrathionate-grown cell suspensions also oxidized thiosulfate at pH 2.0 to 4.0. Apparent Km values (1.2 to 25 mM) were of the same order as for tetrathionate, but kinetics were complex. Continuous culture on growth-limiting tetrathionate at pH 2.5, followed by continuous culture on growth-limiting thiosulfate at pH 2.5, indicated true growth yield values (grams [dry weight] per gram-molecule of substrate) of 12.2 and 7.5, and maintenance coefficient values (millimoles of substrate per gram [dry weight) of organisms per hour) of 1.01 and 0.97 for tetrathionate and thiosulfate, respectively. Yield was increased on both media at low dilution rates by increase in CO2 supply. The apparent maintenance coefficient was lowered without affecting YG, suggesting better energy coupling in CO2-rich environments. Prolonged continuous cultivation on tetrathionate or thiosulfate did not affect the ability of the organism to grow subsequently in ferrous iron medium.     

Thiosulfate and tetrathionate degradation as well as biofilm generation by Thiobacillus intermedius and Thiobacillus versutus studied by microcalorimetry,HPLC, and ion-pair chromatography

The growth of Thiobacillus (T.) intermedius strain K12 and Thiobacillus versutus strain DSM 582 on thiosulfate and tetrathionate was studied combining on-line measurements of metabolic activity and sulfur compound analysis. Most results indicate that T. intermedius oxidized thiosulfate via tetrathionate to sulfate. Concomittantly, sulfur compound intermediates like triand pentathionate were detectable. The formation is probably the result of highly reactive sulfane monosulfonic acids. The formation of tetrathionate allows the cells to buffer temporarily the proton excretion from sulfuric acid production. With T. versutus intermediate sulfur compounds were not detectable, however, sulfur was detectable. The possibility of a thiosulfate oxidation via dithionate, S₂O _inf6 ^sup2- , is discussed. The on-line measurement of metabolic activity by microcalorimetry enabled us to detect that cells of T. intermedius adhere to surfaces and produce a biofilm by a metabolic process whereas those of T. versutus fail to do so. The importance of the finding is discussed.     

Homologs from sulfur oxidation (Sox) and methanol dehydrogenation (Xox) enzyme systems collaborate to give rise to a novel pathway of chemolithotrophic tetrathionate oxidation

The SoxXAYZB(CD)₂‐mediated pathway of bacterial sulfur‐chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock‐out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate‐to‐tetrathionate conversion is Sox independent. Expression of two glutathione metabolism‐related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate‐dependent oxygen consumption pattern of whole cells, and sulfur‐oxidizing enzyme activities of cell‐free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under chemolithotrophic conditions, while expression of a methanol dehydrogenase (XoxF) homolog, subsequently named thiol dehydrotransferase (ThdT), was found to increase 3‐ and 10‐fold during thiosulfate‐to‐tetrathionate conversion and tetrathionate oxidation respectively. A thdT knock‐out mutant did not oxidize tetrathionate but converted half of the supplied 40 mM S‐thiosulfate to tetrathionate. Knock‐out of another thiosulfate dehydrogenase (tsdA) gene proved that both ThdT and TsdA individually converted ～ 20 mM S‐thiosulfate to tetrathionate. The overexpressed and isolated ThdT protein exhibited PQQ‐dependent thiosulfate dehydrogenation, whereas its PQQ‐independent thiol transfer activity involving tetrathionate and glutathione potentially produced a glutathione:sulfodisulfane adduct and sulfite. SoxBCD and SorAB were hypothesized to oxidize the aforesaid adduct and sulfite respectively.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Thiosulfate oxidation and mixotrophic growth of Methylobacterium oryzae

Thiosulfate oxidation and mixotrophic growth with succinate or methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium oryzae CBMB20, which was recently characterized and reported as a novel species isolated from rice. Methylobacterium oryzae was able to utilize thiosulfate in the presence of sulfate. Thiosulfate oxidation increased the protein yield by 25% in mixotrophic medium containing 18.5 mmol.L-1 of sodium succinate and 20 mmol.L-1 of sodium thiosulfate on day 5. The respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfur and sulfite. Thiosulfate was predominantly oxidized to sulfate and intermediate products of thiosulfate oxidation, such as tetrathionate, trithionate, polythionate, and sulfur, were not detected in spent medium. It indicated that bacterium use the non-S4 intermediate sulfur oxidation pathway for thiosulfate oxidation. Thiosulfate oxidation enzymes, such as rhodanese and sulfite oxidase activities appeared to be constitutively expressed, but activity increased during growth on thiosulfate. No thiosulfate oxidase (tetrathionate synthase) activity was detected.     

Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Factors Affecting Oxidation of Thiosalts by Thiobacilli

The effects of temperature, initial pH, and the concentrations of ammonium, phosphate, and heavy metals on the oxidation of thiosalts by an authentic strain of Thiobacillus thiooxidans (ATCC 8085) and by a mixed culture isolated from a base metal-processing mill effluent pond were studied. The optimum temperature was 30°C and the optimum initial pH was 3.75 for both cultures using thiosulfate and for the mixed culture using tetrathionate. T. thiooxidans ATCC 8085 did not oxidize tetrathionate. For a thiosalt concentration of 2,000 ppm (2,000 mg/liter), maximal rates of destruction occurred at concentrations of ammonium ion above 2 mg/liter and in the presence of 1 mg of phosphate per liter. Under optimal conditions, the rate of thiosulfate oxidation by the pure culture was 55 ± 3 mg/liter per h; the mixed culture oxidized thiosulfate at the rate of 40 ± 1 mg/liter per h and tetrathionate at the rate of 50 ± 2 mg/liter per h. Metal ions caused normal inhibition kinetics in the oxidation of thiosulfate by T. thiooxidans ATCC 8085. K_i values were calculated for cadmium (16 mg/liter), copper (0.46 mg/liter), lead (2 mg/liter), silver (3.1 mg/liter), and zinc (33 mg/liter). Only a slight additive effect was apparent in the presence of all of these metal ions. The mixed culture of thiosalt-oxidizing bacteria was less sensitive to heavy metal inhibition; the order of inhibition of thiosulfate oxidation was Cd < Zn < Pb < Ag < Cu, and that of tetrathionate oxidation was Zn < Cd < Pb < Ag < Cu.     

Studies of sulfate utilization by algae 17. Reactions of the adenosine 5'-phosphosulfate (APS) sulfotransferase from Chlorella and studies of model reactions which explain the diversity of side products with thiols

Adenosine 5'-phosphosulfate sulfotransferase has been partiallypurified from Chlorella and is shown to catalyze the transferof the sulfate group of adenosine 5'-phosphosulfate to a varietyof thiol acceptors to form the corresponding organic thiosulfate.While the normal acceptor in the sulfate reducing pathway isthought to be a peptide carrier containing a thiol group theenzyme is very non-specific with respect to the thiols to whichit will transfer leading to a large number of side reactionswhich are possible when thiols are added to the system. Usingadenosine 5'-phosphosulfate and the enzyme, monothiols formsulfite and the organic thiosulfate of the thiol, with dithiolswhich readily form intramolecular disulfides, sulfite is theonly product, while with vicinal dithiols, sulfite and finallythiosulfate is formed. The -SO₃^– sulfur of the thiosulfateoriginates from adenosine 5'-phosphosulfate while the -S^–-sulfur is supplied by the vicinal dithiol. The same productscan be obtained using glutathione-S-sulfonate in place of adenosine5'-phosphosulfate and the enzyme, in a non-enzymatic reactionwith the same thiols. Thus it appears that the enzymatic reactioncatalyzes the transfer of the sulfate group of adenosine-5'-phosphosulfateto a thiol carrier or to any other thiol. When these other thiolsare present, however, sulfite, thiosulfate or organic thiosulfatesof the thiols are formed in non-enzymatic side reactions. Thetransferase from Chlorella is specific for adenosine 5'-phosphosulfateand will not catalyze the reaction with adenosine-3'-phosphate-5'-phosphosulfate. ¹Supported by Grants GB 4321, GB 40856X and BMS 73 00987 AO1from the National Science Foundation. ²Supported by a Gillette Graduate Fellowship. Portions of thispaper formed part of a dissertation presented to the graduatefaculty of Brandeis University in partial fulfillment of thePh.D. Degree. (Received June 30, 1976; )