Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Metabolism of Reduced Inorganic Sulfur Compounds

Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.     

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.     

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.     

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.     

Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans

Tetrathionate is one of the most important intermediates in dissimilatory sulfur oxidation and can itself be utilized as a sole energy source by some sulfur-oxidizing microorganisms. Tetrathionate hydrolase (4THase) plays a significant role in tetrathionate oxidation and should catalyze the initial step in the oxidative dissimilation when sulfur-oxidizing bacteria are grown on tetrathionate. 4THase activity was detected in tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells but not in iron-grown cells. A 4THase having a dimeric structure of identical 50kDa polypeptides was purified from tetrathionate-grown cells. The 4THase showed the maximum activity at pH 3.0 and high stability under acidic conditions. An open reading frame (ORF) encoding the N-terminal amino acid sequence of the purified 4THase was identified by a BLAST search using the database for the A. ferrooxidans ATCC 23270 genome. Heterologous expression of the gene in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. Antisera against the recombinant protein clearly recognized the purified native 4THase, indicating that the ORF encoded the 4THase.     

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.     

Activation of bovine plasminogen by Streptococcus uberis

Abstract Thiosulfate and tetrathionate oxidation activity of Thiobacillus ferrooxidans were found to be absent in iron-growth cell as well as in the cells grown anaerobically on elemental sulfur. While the thiosulfate oxidase activity was absent in the cell-free extract of the above cells, the activity of rhodanese was present irrespective of the culture condition of T. ferrooxidans . It is thus conceivable that rhodanese is not involved in thiosulfate metabolism. During growth in presence of ferrous sulfate plus elemental sulfur, the thiosulfate/tetrathionate oxidation activity was absent till the oxidation of ferrous iron was complete and the cells harvested only in the latter period acquired the thiosulfate/tetrathionate oxidation activity. Thus it becomes evident that the inhibition of thiosulfate and tetrathionate oxidation is solely due to presence of ferrous iron.     

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.     

Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization

Abstract Cell-free extracts of Thiobacillus acidophilus catalysed the stoichiometric conversion of tetrathionate to thiosulphate, sulphur and two protons. The pH optimum of the enzyme activity was 3.0 and its temperature optimum 40°C. The enzyme was unstable at 30 and 40°C, at which its activity decreased to zero within 100 and 20 h, respectively. Enzyme activity was not affected by incubation for 1 week on ice or by freezing and thawing of the extract. The K _m for tetrathionate was 0.3 mM. Enzyme activity was stimulated by ammonium sulphate up to a concentration of 1M. The results indicate that trithionate hydrolase cannot account for the observed conversion of tetrathionate.     

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.     

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.     

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

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

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.     

Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus.

The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N-terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40 degrees C and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions.     

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.     

A Novel Gene Cluster soxSRT Is Essential for the Chemolithotrophic Oxidation of Thiosulfate and Tetrathionate by Pseudaminobacter salicylatoxidans KCT001

Chemolithotrophic sulfur oxidation (Sox) in the α-proteobacterium Pseudaminobacter salicylatoxidans KCT001 was found to be governed by the gene cluster soxSRT–soxVWXYZABCD. Independent transposon-insertion mutations in the genes soxB, soxC, soxD, and also in a novel open reading frame (ORF), designated as soxT, afforded revelation of the entire sox locus of this bacterium. The deduced amino acid sequence of the novel ORF soxT comprised 362 residues and exhibited significant homology with hypothetical proteins of diverse origin, including a permease-like transport protein of Escherichia coli. Two contiguous ORFs, soxR and soxS, immediately preceded the soxT gene. The gene cluster soxSRT was located upstream of soxVWXYZABCD and was transcribed divergently with respect to the latter. Chemolithotrophic utilization of both thiosulfate and tetrathionate was observed to have been impaired in all of these Sox⁻ mutants, implicating the involvement of the gene cluster soxSRT–soxVWXYZABCD in the oxidation of both thiosulfate and tetrathionate. Pradosh Roy Deceased     

Kinetic Enrichment of 34S during Proteobacterial Thiosulfate Oxidation and the Conserved Role of SoxB in S-S Bond Breaking

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of ³⁴S in the end product sulfate, with overall fractionation ranging between −4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of ³⁴S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical ³⁴S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.