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.     

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.     

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.     

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.     

Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus

Arsenical resistance is important to bioleaching microorganisms because these organisms release arsenic from minerals such as arsenopyrite during bioleaching. The acidophile Acidithiobacillus caldus KU was found to be resistant to the arsenical ions arsenate, arsenite, and antimony via an inducible, chromosomally encoded resistance mechanism. Because no apparent alteration of the toxic ions was observed, Acidithiobacillus (At.) caldus was tested to determine if it was resistant as a result of decreased accumulation of toxic ions. Reduced accumulation of arsenate and arsenite by induced At. caldus cells supported this hypothesis. It was also found that, with the addition of an energy source, induced At. caldus could transport arsenate and arsenite out of the cell against a concentration gradient. The lack of efflux in the absence of an added energy source and in the presence of inhibitors suggested that efflux was energy dependent. Induced At. caldus also expressed arsenate reductase activity, indicating that At. caldus has an arsenical resistance mechanism that is analogous to previously described systems from other Bacteria. Southern hybridization analysis showed that At. caldus and other gram-negative acidophiles carry an Escherichia coli arsB homologue on the chromosome.     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

ATP generation during reduced inorganic sulfur compound oxidation by<Emphasis Type="Italic"> Acidithiobacillus caldus</Emphasis> is exclusively due to electron transport phosphorylation

The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus). The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells. The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions. There was no increase in the phosphorylation potential, nor synthesis of ATP. in the absence of electron transport. An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At. caldus. Studies were also performed on the synthesis of ATP by membrane vesicles of At. caldus when presented with an artificial proton gradient. Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors. The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At. caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient.     

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.     

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange

Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.     

A novel Acidimicrobium species in continuous cultures of moderately thermophilic, mineral-sulfide-oxidizing acidophiles

A novel species of Acidimicrobium appeared to be the predominant ferrous iron oxidizer in a mixed culture that effected the continuous, efficient extraction of nickel from a mineral concentrate at 49 degrees C, but it was not isolated in pure culture. It outcompeted Acidimicrobium ferrooxidans, which was expected to have a major role in iron oxidation in reactors gassed with air, and was outnumbered at 49 degrees C only by the sulfur-oxidizing Acidithiobacillus caldus. Sulfobacillus species were expected to compete with Acidimicrobium species when culture aeration was enriched with carbon dioxide, but they were a minor component of the populations with and without this enrichment. Sulfobacillus thermosulfidooxidans replaced the Acidimicrobium species and Acidithiobacillus caldus when the temperature was increased to 55 degrees C.     

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.     

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.     

Low temperature removal of inorganic sulfur compounds from mining process waters

Process water and effluents from mining operations treating sulfide rich ores often contain considerable concentrations of metastable inorganic sulfur compounds such as thiosulfate and tetrathionate. These species may cause environmental problems if released to downstream recipients due to oxidation to sulfuric acid catalyzed by acidophilic microorganisms. Molecular phylogenic analysis of the tailings pond and recipient streams identified psychrotolerant and mesophilic inorganic sulfur compound oxidizing microorganisms. This suggested year round thiosalt oxidation occurs. Mining process waters may also contain inhibiting substances such as thiocyanate from cyanidation plants. However, toxicity experiments suggested their expected concentrations would not inhibit thiosalt oxidation by Acidithiobacillus ferrivorans SS3. A mixed culture from a permanently cold (4-6 °C) low pH environment was tested for thiosalt removal in a reactor design including a biogenerator and a main reactor containing a biofilm carrier. The biogenerator and main reactors were successively reduced in temperature to 5-6 °C when 43.8% of the chemical oxidation demand was removed. However, it was found that the oxidation of thiosulfate was not fully completed to sulfate since low residual concentrations of tetrathionate and trithionate were found in the discharge. This study has demonstrated the potential of using biotechnological solutions to remove inorganic sulfur compounds at 6°C and thus, reduce the impact of mining on the environment.     

The production and utilization of intermediary elemental sulfur during the oxidation of reduced sulfur compounds by Thiobacillus ferrooxidans

The intermediary production of elemental sulfur during the microbial oxidation of reduced sulfur compounds has frequently been reported. Thiobacillus ferrooxidans, an acidophilic chemolithoautotroph, was found to produce an insoluble sulfur compound, primarily elemental sulfur, during the oxidation of thiosulfate, trithionate, tetrathionate and sulfide. This was confirmed by light and electron microscopy. Sulfur was produced from sulfide by an oxidative step, while the production from tetrathionate was initiated by a hydrolytic step, probably followed by a series of chemical reactions. The oxidation of intermediary sulfur was severely inhibited by sulfhydryl-binding reagents such as N-ethylmaleimide, by the addition of uncouplers or after freezing and thawing of the cells, which probably damaged the cell membrane. The mechanisms behind these inhibitions have not yet been clarified. Finally, it was observed that elemental sulfur oxidation by whole cells depended on the medium composition. The absence of sulfate or selenate reduced the sulfur oxidation rate.Non-standard abbreviations NEM N-ethylmaleimide - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.     

Cytotoxic effect of three arsenic compounds in HeLa human tumor and bacterial cells

Numerous epidemiological studies suggest that arsenic (As) compounds are carcinogens, however, recent data have renewed the interest in their anticarcinogenic properties. The cytotoxic effects of three arsenic compounds were assessed: sodium arsenite, sodium arsenate and sodium cacodylate, representing the trivalent and pentavalent species of arsenic, along with a dimethylated pentavalent arsenic species. HeLa cells and Salmonella typhimurium (strains TA98 and TA100) were exposed to As compounds and the cytotoxic effects were evaluated. Alterations on RNA and DNA synthesis in HeLa cells were also examined. All arsenic compounds produced a dose-dependent inhibition on colony formation and DNA synthesis in HeLa cells, yet any of them significantly influenced RNA synthesis in these cells. No evidence of arsenic-induced mutagenicity or antimutagenicity was observed using the Ames assay. In bacterial cells, only sodium arsenite caused a dose-dependent inhibition of colony formation.Collectively, these results indicate that in both, HeLa and S. typhimurium cell systems, only trivalent sodium arsenite can act as an effective inhibitor of cell growth. The possible mechanism(s) of the cytotoxic effect of arsenite in these two different cell systems might be due to its reactivity with intracellular sulfhydryl groups.     

Oxidation of inorganic sulfur compounds by obligatory 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 of 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 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.     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Preparation and purification of As-labeled arsenate and arsenite for use in biological experiments

Inorganic arsenic may occur in biological systems as arsenite or arsenate, these two forms of arsenic differing markedly in both their chemical and biological properties (1). Preparations of arsenic-74 sometimes contain arsenic in both oxidation states. Lunde (2) reported that a sample of ⁷⁴As-labeled sodium arsenate contained 60% of the arsenic-74 as arsenite, while Chan et al. (3) found that labeled arsenate samples contained 0.1 to 1% of an impurity which did not migrate with authentic arsenate during paper electrophoresis.