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.     

Resistance of Thiobacillus novellus to mitomycin C and rifampicin.

Thiobacillus novellus is highly resistant to mitomycin C and rifampicin. This resistance is not due to insusceptibility of the target molecules to the drugs, since mitomycin C cross-links the DNA and rifampicin inhibits the DNA-dependent RNA polymerase of T. novellus in vitro.     

Growth and physiology of Thiobacillus novellus under nutrient-limited mixotrophic conditions.

Thiobacillus novellus was cultivated in a chemostate under the individual limitations of thiosulfate, glucose, and thiosulfate plus glucose. At dilution rate (D) of 0.05 h-1 or lower, the steady-state biomass concentration in mixotrophic medium was additive of the heterotrophic and autotrophic biomass at corresponding D values. The ambient concentrations of thiosulfate, glucose, or both in the various cultures were low and were very similar in mixotrophic, heterotrophic, and autotrophic environments at a given D value. At D = 0.05 h-1, mixotrophic cells possessed higher activities of sulfite oxidase and thiosulfate oxidation compared to autotrophic cells, as well as higher activities of glucose enzymes and glucose oxidation than heterotrophic cells. Thus, in contrast to nutrient-excess conditions, in nutrient-limited mixotrophic environments at these D values, T. novellus did not exhibit characteristics of uncoupled substrate oxidation, inhibition of substrate utilization, and repression of enzymes of energy metabolism. It is concluded that T. novellus responds to mixotrophic growth conditions differently in environments of different nutritional status, and the ecological and physiological significance of this finding is discussed.     

Arsenic toxicity is not due to a direct effect on the oxidation of reduced inorganic sulfur compounds by Thiobacillus caldus

Abstract Thiobacillus caldus is a moderately thermophilic acidophile which has been implicated in the biooxidation of arsenic containing mineral Sulfides. The toxic effects of arsenic on this bacterium are presented here. Addition of arsenite to a growing culture of T. caldus caused a transient increase in the optical density of the culture while causing a simultaneous decrease in cell viability. The increase in optical density was shown to be due to the formation of extracellular sulfur. The oxidation rates of tetrathionate and thiosulfate were decreased by increasing concentrations of arsenite, while in a culture induced to arsenic resistance the rates were not as adversely effected. Sulfur oxidation was also inhibited to the same extent as tetrathionate oxidation, with the oxidation of solid sulfur being slightly more effected than the oxidation of sulfur dissolved in acetone. Thus, bactericidal arsenite causes a transient formation of extracellular sulfur in the culture supernatant of T. caldus yet the toxicity of arsenite is not due to direct inhibitory effects on reduced inorganic sulfur compound oxidation by these bacteria.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Competition between the facultatively chemolithotrophic Thiobacillus A2, an obligately chemolithotrophic Thiobacillus and a heterotrophic spirillum for inorganic and organic substrates

Competition in a chemostat between the versatile Thiobacillus A2 and the specialized T. neapolitanus for thiosulfate as the sole growth-limiting substrate, led to dominance of the specialized over the versatile organism, at dilution rates 0.025 h^-1. Increasing concentrations of acetate or glycollate in the thiosulfate medium caused increased relative numbers of T. A2 in steady states at D=0.07 h^-1. Eventually, with 10–12 mmol of organic substrate per litre, complete dominance of T. A2 over T. neapolitanus occurred.Mixed cultures of T. A2 and a specialized spirillumshaped heterotroph, competing for acetate as sole growth-limiting substrate resulted in complete dominance of the heterotroph at dilution rates of 0.07 and 0.15 h^-1. In this case increasing concentrations of thiosulfate in the acetate medium, up to 10 mM, eventually led to the elimination of the heterotroph.These results have been interpreted as evidence that T. A2 was growing mixotrophically. As the concentration of the second substrate was raised, the number of T. A2 cells increased and as a result T. A2 consumed an increasing portion of the common substrate.In mixed chemostat cultures containing all three organisms, T. A2 could maintain itself with all tested ratios of acetate and thiosulfate in the inflowing medium. The heterotroph was excluded from the culture below a relatively low acetate to thiosulfate ratio, whilst above a relatively high acetate to thiosulfate ratio T. neapolitanus was completely eliminated.These results were discussed in relation to the ecological niche of Thiobacillus A2-type organisms.     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.     

Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans.

An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.     

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.     

The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase. ATP affects the oxidase uniquely.

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady-state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]-v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis-Menten-type hyperbola in the presence of 700 microM ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 microM ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 microM ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]-v curve and the molecular mass of the oxidase when used in place of ATP.     

A comparison of plant communities on the basis of their clonal growth patterns

This study was performed to analyse how a vegetative propagation pattern of plants affects the coexistence of species and subsequent species richness of the community. We compared community average clonal growth in the herbal communities of forests, wooded meadows, and open meadows in Laelatu, Estonia. The parameters used for the calculation of the community averages and measured for each species were ramet life span, rhizome branching, and clonal mobility. We also examined the intrinsic (i.e. independent of the environment) relationship between community clonal growth and plant species density. We found strong correlations between the environmental factors (productivity, light availability, and mowing regime) and community averages of clonal growth parameters, while species density was (negatively) correlated only with community average of rhizome increment. The community average of ramet life span decreased with the increasing biomass of the herb layer. No evidence was found to support the hypothesis that species-rich communities may consist of species with more contrasting mobility compared with species-poor communities. Independent of the effect of the environmental factors, species density was positively correlated with ramet density. There was intrinsic positive relationship between species density and community average of ramet life span at open meadow sites and intrinsic negative relationship between species density and community average of rhizome increment at wooded meadow sites. We conclude that in forest communities the capability of clonal plants to forage for light is favoured, while in unmown meadows a competitively strong phalanx growth form is advantageous. We established that ramet turnover increases and vegetative mobility decreases with increasing species diversity, although these two relationships depend strongly on the type of the studied community.Co-ordinating editor: J. Tuomi     

Growth and respiratory oxidation of reduced sulfur compounds by intact cells ofThiobacillus novellus (type strain) grown on thiosulfate

Thiobacillus novellus (type strain) was grown chemolithoautrophically on thiosulfate in batch cultures under microaerophilic conditions. Under these conditions,T. novellus grew exponentially (=0.05–0.06 h^–1). The respiratory oxidation rates of tetrathionate, thiosulfate, elemental sulfur (S^o), and sulfite were measured respirometrically with an oxygen electrode, with exponentially growing cells. Cells growing on thiosulfate as the unique energy source retain thiosulfate-oxidizing activity, S^o-oxidizing activity (SOA), and very high sulfite-oxidizing activity, but lack respiratory tetrathionate-oxidizing activity. HQNO (50 m), an inhibitor of the quinone-cytochrome b region, strongly inhibited the SOA (70%), moderately the sulfite-oxidizing activity (45%), and poorly the thiosulfate-oxidizing activity (15%), 1mm KCN totally inhibited (>89%) all respiratory activities. This study confirms that inThiobacillus novellus, as well as in otherThiobacilli, SOA is present in cells grown with thiosulfate as sole electron donor. SOA appears not to be an oxygenase; it is linked to the respiratory chain, and the electrons are probably released in the quinone-cytochrome b region.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

Identification of two outer membrane proteins involved in the oxidation of sulphur compounds in Thiobacillus ferrooxidans

Abstract: Shift of three Thiobacillus ferrooxidans strains from Fe(II) to S⁰ or thiosulphate liquid medium caused distinctive changes in the outer membrane protein profile. In addition to a new 55-kDa protein which was synthesized only in the presence of sulphur compounds, a higher expression of a 47-kDa protein was observed. This latter protein appeared to be constitutively synthesized, since it was detectable in small amounts even in tile presence of ferrous iron as sole energy source, but its expression was greatly enhanced when elemental sulphur or thiosulphate were present in the growth medium.