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PubMed1996

NMR assignments and relaxation studies of Thiobacillus versutus ferrocytochrome c-550 indicate the presence of a highly mobile 13-residues long C-terminal tail.

Cytochrome c-550 of Thiobacillus versutus functions as an electron transfer protein in a chain of redox proteins that enables T. versutus to grow on methylamine. It is a single-heme protein of 134 residues, related to mitochondrial cytochrome c. Cytochrome c-550, as well as several other bacterial c2-type cytochromes, contain a C-terminal extension of 13-16 amino acids of unknown function, compared to mitochondrial cytochrome c. NMR experiments were performed to obtain structural and dynamic information on the protein in solution. For this purpose, T. versutus cytochrome c-550 was labeled with 15N and 13C using 13C-methanol grown Paracoccus denitrificans as a host for heterologous expression. NMR assignments were obtained for the 1H, 15N, and 13C nuclei in the backbone and the beta-positions of the protein and the secondary structure was determined. 15N-relaxation studies were performed to characterize the dynamic properties of the protein. The results indicate that the main part of T. versutus ferrocytochrome c-550 exists in solution as a rigid, well-ordered molecule with a secondary structure that is very similar to that of P. denitrificans cytochrome c-550, as observed in crystals. The C-terminal extension, however, is unstructured and highly mobile. The possible origin and function of the extension are discussed.

Amino Acid Sequence↗

PubMed1994

Active site geometry in the high oxido-reduction potential rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a blue copper protein involved in the oxidation of iron catalyzed by Thiobacillus ferrooxidans. This protein is characterized by a high oxido-reduction potential and a high stability at low pH. The three dimensional structure of this protein is still unknown and in order to investigate the geometric properties of the copper center which could be correlated to the high oxido-reduction potential, we have studied rusticyanin by UV-Visible, EPR and NMR spectroscopies, at different pH values. Our results suggest that rusticyanin is stable between pH 2 and pH 9 and that the copper center does not undergo significant geometric modifications in this pH range. Moreover, the copper atom could be buried more deeply in the protein than in other type I copper proteins and the atomic distance Cu-S(Met), one of the four bonds involved in copper coordination, is probably shorter in rusticyanin than in other cupredoxins. These two properties of the copper site are expected to be responsible, in part, for the high oxido-reduction potential observed in rusticyanin.

Azurin↗

PubMed1994

Solution structure of the type 1 blue copper protein amicyanin from Thiobacillus versutus.

A three-dimensional solution structure of amicyanin from Thiobacillus versutus has been determined by distance geometry and restrained molecular dynamics. A total of 984 experimentally derived constraints were used for the final refinement (881 distance constraints and 103 dihedral angle constraints). Stereospecific assignments were made for 17 prochiral beta-methylene protons (33%) and the methyl groups of eight valine residues. Fourteen structures were selected to represent the solution structure. They show an average pairwise backbone root-mean-square deviation of 1.19 A. The overall structure can be described as a beta-sandwich, built up of nine beta-strands. The copper atom is located between three loops on one end of the molecule. Two of these loops contribute the copper ligands. His54 is on the loop between beta-strands 4 and 5. The other three ligands, Cys93, His96 and Met99, are located evenly spaced on the loop between beta-strands 8 and 9. This loop is folded in two consecutive type 1 turns with His96 as the donor and acceptor of the NHi-CO(i-3) hydrogen bonds. The folding is reminiscent of the general cupredoxin fold. Considerably different are the large 21 residue N-terminal extension, that is unique to amicyanin and forms an extra beta-strand (strand 1), and the region between beta-strands 5 and 7. The partly surface-exposed copper ligand His96 is surrounded by a hydrophobic patch consisting of seven residues.

Bacterial Proteins↗

PubMed1996

NMR solution structure of Cu(I) rusticyanin from Thiobacillus ferrooxidans: structural basis for the extreme acid stability and redox potential.

The solution structure of the Cu(I) form of the rusticyanin from Thiobacillus ferrooxidans has been calculated from a total of 1979 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal two beta-sheets, one of six strands and one of seven strands that are tightly packed in a beta-barrel or beta-sandwich arrangement, and a short helix that extends on the outside of one of the sheets to form a second hydrophobic core. The copper coordination sphere is composed of the standard type I ligands (His2CysMet) in a distorted tetrahedral arrangement. The copper-binding site is located within a hydrophobic region at one end of the molecule, surrounded by a number of aromatic rings and hydrophobic residues. This configuration probably contributes to the acid stability of the copper site, since close association of the aromatic rings with the histidine ligands would sterically hinder their dissociation from the copper. An electrostatic analysis based on a comparison of the structures of rusticyanin and French bean plastocyanin shows that factors determining the high redox potential of rusticyanin include contributions from charged side-chains and from the disposition of backbone peptide dipoles, particularly in the 81 to 86 region of the sequence and the ligand cysteine residue. These interactions should also contribute to the acid stability by inhibiting protonation of His143.

Azurin↗

PubMed1995

Isolation and characterization of the replicon of a Thiobacillus intermedius plasmid.

The replicon of a cryptic Thiobacillus intermedius plasmid (pTiK12) has been isolated and sequenced. Functional analysis of deletion subclones in Escherichia coli localized the replicon to a 3.5-kb region of DNA. Sequencing of this region identified a 30-bp A-T-rich potential stem-loop structure. In addition, an 11-bp direct repeat, an 11-bp inverted repeat, and a 16-bp inverted repeat were observed at the stem-loop structure. Also found in the replicon was a series of four tandem direct repeats consisting of a perfectly conserved 8-bp core. A region near the stem-loop structure is involved in the regulation of plasmid copy number. Deletion subclones lacking this region have increased copy numbers, indicating a negative regulatory role. An open reading frame capable of encoding a 320-amino-acid protein was found near the stem-loop structure. The putative amino acid sequence shares significant similarity with the two Rep proteins from the ColE2 and ColE3 replicons. Replication of the T. intermedius replicon is dependent upon DNA polymerase I. The isolation and examination of the T. intermedius plasmid replicon are initial steps toward the establishment of a genetic system in T. intermedius.

Algorithms↗

PubMed1997

Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans.

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans. The 1.4-kb fragment hybridized with IST2, an insertion sequence of T. ferrooxidans. The 1.2-kb fragment was cloned and sequenced. The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with ISAE1 of Alcaligenes eutrophus. The IST445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T. ferrooxidans. In Southern blots of restricted genomic DNAs probed with IST445, each of the several strains of T. ferrooxidans gives a distinctive fingerprint. IST445 is present in the range of 10-20 copies per genome in the four strains studied.

Amino Acid Sequence↗

PubMed1998

Sequence analysis of plasmid pTF5, a 19.8-kb geographically widespread member of the Thiobacillus ferrooxidans pTFI91-like plasmid family.

The most important features of plasmid pTF5, a 19,793-bp plasmid that was isolated from Thiobacillus ferrooxidans ATCC33020, are presented. The plasmid contained at least 14 complete open reading frames (ORFs), most of which had clear amino acid sequence similarity to previously identified proteins. The majority of ORFs were related to proteins commonly found on plasmids such as replication-, partition-, and stability-associated proteins or on transposons such as transposases, an invertase, and a resolvase. Products of three of the ORFs were related to redox-active proteins and possibly constitute an electron transport system. Plasmids with restriction endonuclease maps identical to that of pTF5 have been widely reported among T. ferrooxidans strains, and in this study, pTF5 was itself shown to be a member of the widely distributed pTFI91 T. ferrooxidans plasmid family. A comparison of restriction endonuclease maps indicated that a pTFI91-like plasmid (9.8 kb) appeared to be contained entirely within pTF5.

Amino Acid Sequence↗

PubMed2000

pT3.2I, the smallest plasmid of Thiobacillus T3.2.

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3. 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.

Amino Acid Sequence↗

PubMed1990

Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite, arsenate, ferrous and ferric iron.

Two strains of Thiobacillus, T. ferrooxidans and T. thiooxidans, have been isolated from a bacterial inoculum cultivated during a one-year period in a 1001 continuous laboratory pilot for treatment of an arsenopyrite/pyrite concentrate. The optimum pH for the growth of both strains has been found to be between 1.7 and 2.5. Because of the high metal toxicity in bioleach pulps, the tolerance of T. ferrooxidans and T. thiooxidans with respect to iron and arsenic has been studied. The growth of both strains is inhibited with 10 g/l of ferric ion, 5 g/l of arsenite and 40 g/l of arsenate. 20 g/l of ferrous iron is toxic to T. ferrooxidans but 30 g/l is necessary to impede the growth of T. thiooxidans.

Acidithiobacillus thiooxidans↗

PubMed1976

Energetic aspects of the metabolism of reduced sulphur compounds in Thiobacillus dentrificans.

Yields of Thiobacillus dentrificans on different substrates were compared. The organism was grown in a chemostat at a dilution rate of 0.03 h-1. From the difference in the cell yields with (1) oxygen (6.40 g carbon per mole thiosulphate) and (2) nitrate (4.51 g carbon per mole thiosulphate) as an electron acceptor the experimental value for Y ATP was estimated to be 1.75. The efficiency of the biosynthetic system would be 42% if 1 ATP should be needed in reversed electron transport, and 57% if this was 2 ATP per electron pair. It could be calculated that during anaerobic oxidation of thiosulphate with nitrate 1.41 or 1.16 ATP per 2 electrons are generated if 1 or 2 ATP respectively per thiosulphate is formed in substrate-level phosphorylation. For aerobic oxidation these figures are 2.40 and 2.16, respectively.

Aerobiosis↗

PubMed1980

Substrate level versus oxidative phosphorylation in the generation of ATP in Thiobacillus denitrificans.

Particulate fractions of Thiobacillus denitrificans catalyse that the phosphorylation of ADP to ATP during the oxidation of various inorganic sulphur compounds or NADH via an electron transport chain. On the other hand, a "soluble" cell-free fraction synthesized ATP from APS and inorganic phosphate. The production of ATP was verified either by the firefly luciferin-luciferase enzyme system or by the incorporation of 32Pi into ATP. During the oxidation of sulphide, sulphite and NADH the production of ATP from ADP by particulate fractions is inhibited by compounds that inhibit electron transfer and by uncouplers of oxidative phosphorylation. However, these compounds had little effect on the production of ATP from AMP during the oxidation of sulphite by the soluble fraction. NADH was the most effective electron donor for oxidative phosphorylation. The soluble fraction contained high activities of ATP sulphurylase, inorganic pyrophosphatase and adenylate kinase but ADP sulphurylase activity was relatively low. The effects of inhibitors on ATP production from APS and Pi are compared with those on adenylate kinase and ATP sulphurylase.

Adenosine Diphosphate↗

PubMed1986

Kinetics and energetics of reduced sulfur oxidation by chemostat cultures of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans was grown in chemostat cultures with thiosulfate and tetrathionate as the limiting substrates. The yields at steady state on both substrates at different dilution rates were calculated. In a few experiments the air supply was supplemented with 2% CO2 (v/v). This resulted in a slightly increased yield. Cells from the chemostat cultures were used to study the kinetics of thiosulfate, tetrathionate, sulfite and sulfide oxidation. With all substrates mentioned the Ks values were in the micromolar range. The values for thiosulfate and tetrathionate were 2 orders of magnitude lower that those published previously.

Kinetics↗

PubMed1977

Proton translocation in intack cells of Thiobacillus denitrificans.

Proton translocation assessed by the quinacrine fluorescence technique was compared with oxygen uptake during thiosulphate oxidation by cells of Thiobacillus denitrificans. The addition of thiosulphate to cell suspensions resulted in an outwardly directed proton translocation as reflected by an increased quinacrine fluorescence. Compared to the O2 uptake activity, the proton translocating system was much more sensitive to proton conductors, other ionophores and inhibitors of electron transport. The results indicate that (a) the proton-translocation activity (membrane energization) is enhanced in aged cell suspensions, (b) intactness of the cytoplasmic membrane is essential for establishing a protonmotive force in cells, (c) the fluorescence increase and proton translocation are reversible processes, (d) inhibitors of electron transport may also act as proton conductors by altering the integrity of the cytoplasmic membrane.

Cell Membrane↗

PubMed1986

Chemostat enrichment and isolation of Hyphomicrobium EG. A dimethyl-sulphide oxidizing methylotroph and reevaluation of Thiobacillus MS1.

A stable mixed bacterial culture was obtained by chemostat enrichment using dimethyl-sulphoxide as a carbon and energy source. This culture could not only rapidly oxidize dimethyl-sulphoxide but also dimethyl-sulphide. Enzyme determinations indicated that an important part of it consisted of methylotrophs, which assimilated carbon via the serine pathway. Indeed plate counts revealed the majority of the community to be a Hyphomicrobium species. This organism, designated Hyphomicrobium EG, is an obligate methylotroph which can only grow aerobically on several different C1-compounds. Its performance on dimethyl-sulphoxide was compared with that of the community and of another recently isolated strain, Hyphomicrobium S. The mixed culture, Hyphomicrobium EG and Hyphomicrobium S had a mu max of 0.08, 0.08 and 0.014 h-1 respectively. The KS for dimethyl-sulphoxide was the same for all three cultures (3-6 microM), whereas that for dimethyl-sulphide of Hyphomicrobium EG after growth on dimethyl-sulphoxide was 3-fold higher than that of the other two cultures (48 and 16 microM respectively). After growth on dimethyl-sulphide it improved to 3 microM. Dimethyl-sulphide respiration was maximal at a concentration of 100 microM; higher concentrations were inhibitory. One of the accompanying organisms, a pink methylotroph, was able to derive energy from the oxidation of thiosulphate. Available cultures of Thiobacillus MS1 that were reported to be able to utilize dimethyl-sulphide could no longer metabolize this compound.

Bacteria↗

PubMed1985

The major soluble cytochromes of the obligately aerobic sulfur bacterium, Thiobacillus neapolitanus.

Four cytochromes were isolated from soluble extracts of the aerobic sulfur bacterium, Thiobacillus neapolitanus. The two most abundant proteins were purified to homogeneity and thoroughly characterized. Cytochrome c-554 (547) is a monomeric, small molecular weight protein which is unusual in having two well-resolved alpha peaks in UV-visible absorption spectra. The redox potential is 208 mV. Native cytochrome c-549 is oligomeric, but has a subunit size of about 26,000. The yield of this protein could be improved dramatically by washing membranes with 30% ammonium sulfate, but the material solubilized by this method had a larger native molecular weight than that in the initial 0.1 M Tris-Cl extract and behaved differently on chromatography. The properties of cytochrome c-549 including subunit size and UV-visible absorption spectra are similar to mitochondrial cytochrome c1 and chloroplast cytochrome f, which suggests that it may be a modified form of the predominant membrane cytochrome. Based on cytochrome content, it is suggested that T. neapolitanus is not closely related to other thiobacilli.

Amino Acids↗

PubMed1976

Ribulose biophosphate carboxylase from Thiobacillus A2. Its purification and properties.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an Srel. of 16.9. Km for RuBP was 122 muM, for total "CO2" it was 4.17 mM, and for Mg2+ 20.0 muM. The absolute requirement for a divalent cation was satisfied by Mg2+ which was replaceable to a certain extent by Mn2+. Activity was not significantly affected by SO(2-4), SO(2-3), or S(2)O(2-3) at 1.0 mM. At this concentration S(2-) caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.04 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a Ki of 20 muM. Lost activity could be restored partially by GSH or Cu2+. Although azide at the concentration tested had no significant effect on enzyme activity, 2, 4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.

Adenine Nucleotides↗

PubMed1977

Acidostability of speroplasts prepared from Thiobacillus thiooxidans.

Thiobacillus thiooxidans was acidostable even in the absence of its respiratory substrate, elementary sulfur. This suggests that the acidostability of the bacterium was enery-independent. The organism was subjected to osmotic shock with 0.75 M sucrose at 0 degrees C and then treated with snail intestinal juice in the presence of 0.3 M sucrose. The decrease in the optical density of the sample thus prepared on dilution with deionized water and electron microscopic observation of the sample showed that spheroplasts were formed from the bacterium by this procedure. Spheroplasts were able to respire sulfur and their respiratory activity was acidostable. Spheroplasts, when treated with Nagase, proteolytic enzyme, lost their acidostability, and some protein components disappeared from the membrane fraction. This suggests that the acidostability of the bacterium may be related to protein conponents of the membrane.

Acidithiobacillus thiooxidans↗

PubMed1976

Utilization of 35S-thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans.

Differentially labelled 35S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5-4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated Km value of 0.5 mM for 35S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50 degrees C. In the absence of thiosulphate, the cells bound 35S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-35S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-35S]thiosulphate or 35S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP-sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.

Ferrous Compounds↗