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Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences.

5S rRNA nucleotide sequences from Thiobacillus neapolitanus, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus intermedius, Thiobacillus perometabolis, Thiobacillus thioparus, Thiobacillus versutus, Thiobacillus novellus, Thiobacillus acidophilus, Thiomicrospira pelophila, Thiomicrospira sp. strain L-12, and Acidiphilium cryptum were determined. A phylogenetic tree, based upon comparison of these and other related 5S rRNA sequences, is presented. The results place the thiobacilli, Thiomicrospira spp., and Acidiphilium spp. in the "purple photosynthetic" bacterial grouping which also includes the enteric, vibrio, pseudomonad, and other familiar eubacterial groups in addition to the purple photosynthetic bacteria. The genus Thiobacillus is not an evolutionarily coherent grouping but rather spans the full breadth of the purple photosynthetic bacteria.

Base Sequence↗

Preliminary proteomic analysis of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately.

Thiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe2+ or sulphide as energy source. Growth curves for Thiobacillus ferrooxidans have been tested, which show lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Thiobacillus ferrooxidans cultivated with Fe2+ and from 4 to 12 days for Thiobacillus ferrooxidans cultivated with elemental sulphur. Differences of protein patterns of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately were investigated after cultivation at 30 degrees C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ ionization (MALDI)-Mass spectrometry and ESI-MS/MS. From the 17 identified protein spots, 11 spots were found more abundant when growing on elemental sulphur. By contrast 6 protein spots were found decreased at elemental cultivation condition. Among the proteins identified, cytochrome C have been previously identified as necessary elements of electron-transferring pathway for Thiobacillus ferrooxidans to oxidize Fe2+; ATP synthase alpha chain and beta are expressed increased when Thiobacillus ferrooxidans cultivated with Fe2+ as energy source. ATP synthase Beta chain is the catalytic subunit, and ATP synthase alpha chain is a regulatory subunit. The function of ATPase produces ATP from ADP in the presence of a proton gradient across the membrane.

ATP Synthetase Complexes↗

Thiobacillus plumbophilus spec. nov., a novel galena and hydrogen oxidizer.

From an uranium mine three strains of rod-shaped, mesophilic, chemolithouautotrophic bacteria were isolated. They grow by oxidation of H2S, galena (PbS) and H2. Angelsite (PbSO4) is formed from galena. No ferrous iron is oxidized by the isolates. They grow between pH 4 and 6.5 at temperatures of about 9 to 41 degrees C (optimum around 27 degrees C). The G + C content of the DNA is around 66 mol %. Based on their ability to oxidize sulfur compounds, the new organisms belong to the genus Thiobacillus. No significant homology with Thiobacillus ferrooxidans and Thiobacillus cuprinus was detected by DNA-DNA hybridization. Therefore the new isolates represent a new species within the genus Thiobacillus. Based on the unusual growth on galena, we name the new species Thiobacillus plumbophilus (type strain Gro7; DSM 6690).

Base Composition↗

Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans.

From cell yields of Thiomicrospira denitrificans grown inthe chemostat at different growth rates under anaerobic conditions a value of 1.4 mM S2O3 = per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 dry wt per mole S2O3 = for the true growth yield. Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans. Though in Thiobacillus denitrificans at D = 0.03 h(-1) under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans. Under aerobic conditions at D = 0.03 h(-1) values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate. As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate. Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans. This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase.

Adenosine Triphosphate↗

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral.

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans. Pyrite (FeS2) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe2+), elemental sulphur (S0) or FeS2. Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe2+ and S0 oxidation, NaN3 and NEM, respectively, partially abolished FeS2 oxidation. b. A b-type cytochrome was detectable in FeS2- and S0-grown cells but not in Fe2+-grown cells. c. FeS2 and S0 reduced b-type cytochromes in whole cells grown on S0. d. CO2 fixation at pH 4.0 per mole of oxygen consumed was the highest with S0, lowest with Fe2+ and medium with FeS2 as substrate. e. Bacterial Fe2+ oxidation was found to be negligible at pH 5.0 whereas both FeS2 and S0 oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe3+ was not affected by separation of pyrite and bacteria. Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.

Azides↗

Purification and characterization of a periplasmic Thiosulfate dehydrogenase from the obligately autotrophic Thiobacillus sp. W5.

A periplasmic thiosulfate dehydrogenase (EC 1.8.2.2) was purified to homogeneity from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5. A five-step procedure resulted in an approximately 2,300-fold purification. The purified protein had a molecular mass of 120 +/- 3 kDa, as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 33 +/- 1 kDa and 27 +/- 0.5 kDa, as determined by SDS-PAGE. UV/visible spectroscopy revealed that the enzyme contained haem c; haem staining showed that both subunits contained haem c. A haem c content of 4 mol per mol of enzyme was calculated using the pyridine haemochrome test. The pH optimum of the enzyme was 5.5. At pH 7.5, the Km and Vmax were 120 +/- 10 microM and 1,160 +/- 30 U mg-1, respectively. The absence of 2-heptyl-4-hydroquinoline-N-oxide (HQNO) inhibition for the oxidation of thiosulfate by whole cells suggested that the electrons enter the respiratory chain at the level of cytochrome c. Comparison with thiosulfate dehydrogenases from other Thiobacillus species showed that the enzyme was structurally similar to the thiosulfate dehydrogenase of the acidophilic, facultatively chemolithoautotrophic Thiobacillus acidophilus, but not to the thiosulfate dehydrogenases published for the obligately chemolithoautotrophic Thiobacillus tepidarius and Thiobacillus thioparus.

Bacterial Proteins↗

Phylogeny of Thiobacillus cuprinus and other mixotrophic thiobacilli: proposal for Thiomonas gen. nov.

The complete 5S and 16S ribosomal DNA (rDNA) sequences of the facultatively chemolithotrophic bacterium Thiobacillus cuprinus and results of a comparison of these sequences with homologous sequences from several proteobacterial species supported affiliation of T. cuprinus with the beta 1 subgroup of the Proteobacteria. T. cuprinus, Thiobacillus intermedius, Thiobacillus perometabolis, and Thiobacillus thermosulfatus form a phylogenetic cluster that comprises some of the thiobacilli capable of mixotrophic growth. This cluster is related to some pseudomonads and Alcaligenes species belonging to the beta subclass. In addition, a low-frequency restriction fragment analysis (LFRFA) of some mixotrophic thiobacilli and some related species was carried out by using pulsed-field gel electrophoresis (PFGE) to determine the SpeI and XbaI macrorestriction patterns and genome sizes of these organisms. The correlation of the LFRFA results and the 16S rDNA analysis results and the usefulness of the two analyses are discussed. The PFGE fingerprints suggested that Thiobacillus sp. strain ATCC 27793 is related to T. intermedius rather than to T. perometabolis, as described previously. The distinctive characteristics of the mixotrophic species analyzed in this work, their phylogenetic relatedness, and their physiological differences from other groups belonging to the Proteobacteria, including other thiobacilli, suggest that these organisms should be transferred to a new genus, the genus Thiomonas gen. nov.

DNA, Bacterial↗

[Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khoito-Gol hydrogen-sulfide springs, Buryatia].

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8-9.5, with an optimum at pH 9.3 and in a temperature range of 5-39 degrees C, with an optimum at 28-32 degrees C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3-64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21-29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).

Base Composition↗

Thiobacillus cuprinus sp. nov., a Novel Facultatively Organotrophic Metal-Mobilizing Bacterium.

Five strains of mesophilic, facultatively organotrophic, ore-leaching eubacteria were isolated from solfatara fields in Iceland and a uranium mine in the Federal Republic of Germany. The new organisms are aerobic gram-negative rods. They can use sulfidic ores or elemental sulfur as sole energy source, indicating that they belong to the genus Thiobacillus. Alternatively, they grow on organic substrates such as yeast extract, peptone, and pyruvate. In contrast to the other leaching bacteria known so far, the new isolates are unable to oxidize ferrous iron. They consist of extreme and moderate acidophiles growing optimally at pH 3 and 4, respectively. The extreme acidophiles showed leaching characteristics similar to those shown by Thiobacillus ferrooxidans, while the moderate acidophiles exhibited a pronounced preference for copper leaching on some chalcopyrite ores. The G+C content of the DNA is between 66 and 69 mol%, depending on the isolate. In DNA-DNA hybridization experiments, the new strains showed homologies among each other of >70%, indicating that they belong to the same species. No significant DNA homology to Thiobacillus reference strains was detectable. Therefore, the new isolates represent a new species of Thiobacillus, which we name Thiobacillus cuprinus. Isolate Hö5 is designated as the type strain (DSM 5495).

Journal Article↗

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2.

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Duodoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with 14C in carbon atoms 1, 2, 3, 3 + 4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Duodoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.

Arsenic↗

Effect of sulphur inoculated with Thiobacillus on soil salinity and growth of tropical tree legumes.

A greenhouse experiment was carried out with the objective of evaluating the effects of the elementary sulphur inoculated with Thiobacillus, compared with gypsum, in the amendment of a alluvial sodic saline soil from the Brazilian semiarid region, irrigated with saline water and grown with the tropical legumes leucena and mimosa. The treatments consisted of levels of sulphur (0; 300 and 600 kg/ha) and gypsum (1,200 and 2,400 kg/ha), irrigation using different waters containing the salts NaHCO3, MgCl2, CaCl2, NaCl and KCl, with different electrical conductivities (ECs: 0.2. 6.1 and 8.2 dS/m at 25 degrees C). Based on the results it appears that saline water increased exchangeable Na+, K+, Ca2+, Mg2+, and soil pH. Sulphur inoculated with Thiobacillus was more efficient than gypsum in the reduction of the exchangeable sodium of the soil and promoting leaching of salts, especially sodium. Sulphur inoculated with Thiobacillus reduced the EC of the soil saturation extract to levels below that adopted in soil classification of sodic or saline sodic. Leucena was more tolerant to salinity and mimosa more resistant to acidity promoted by sulphur inoculated with Thiobacillus.

Brazil↗

Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov.

The species of the genus 'Thiobacillus' fall into the alpha-, beta- and gamma-subclasses of the Proteobacteria, the type species Thiobacillus thioparus being located in the beta-subclass. 'Thiobacillus' species exhibit almost as much diversity in DNA composition and physiology as is found collectively in all other proteobacterial groups. On the basis of physiological characters and 16S rRNA gene sequence comparisons, eight of the existing Thiobacillus species are proposed for reassignment to three newly designated genera within the gamma-subclass of the Proteobacteria, namely Acidithiobacillus, Halothiobacillus and Thermithiobacillus.

Genes, rRNA↗

Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli.

Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+. (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells. The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358. Both systems, cloned and expressed in E. coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion. Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC. Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA. Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC. Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.

Biological Transport↗

[Cloning and orientation of a promoter of thermophilic Thiobacillus sp].

Thiobacillus sp. is an obligate autotrophic thermophilic bacterium which was isolated from an acidic hot spring in Yunnan Province. Its optimum growth temperature is 45-50 degrees C and its optimum pH is 2.0-3.0. Using DNA recombinant technique, we inserted the HindIII fragments of the Thiobacillus sp. chromosomal DNA into the HindIII site of promoter-probe plasmid pSDSI (AprTcs, 5.65 kb). Transformants resistant to tetracycline were obtained on Tc plates (12 micrograms/ml). Of these, twenty transformants were able to grow on 120 micrograms/ml Tc plates, and two of them, designated pSDH7 and pSDH11, were able to grow on plates containing Tc at concentration up to 360 micrograms/ml. With HindIII, pSDH11 produced a 0.95kb fragment which had the function of promoter and a PstI site besides the 5.65 kb fragment of pSDSI. Southern blot hybridization showed that the 0.95 kb insert was from the Thiobacillus sp. chromosomal DNA. After restriction mapping, a 2.85 kb fragment of pSDH11 (which contained 0.7 kb of the inserted fragment) was removed with the aid of Pst1, and the remained fragment was used to construct a 3.75 kb plasmid (named pSDH114) which was resistant to a higher level of tetracycline (360 micrograms/ml) than for pBR322 (120 micrograms/ml). The remained 0.25 kb foreign fragment in pSDH114 still retained full function of the promoter contained in the original 0.95 kb. Thus we could orient the cloned promotor function fragment (0.25 kb) from Thiobacillus sp. in pSDH114.

Cloning, Molecular↗

Wide-host-range plasmids function in the genus thiobacillus.

Plasmids S-a, RP4, R388, and several RP4 derivatives (pMD101, pDT387, and pDT566) were transmissible by conjugation to Thiobacillus novellus from Escherichia coli. Genetic markers were expressed in T. novellus, with the exception of chloramphenicol resistance and ampicillin resistance. Plasmids were not transmissible by conjugation from E. coli donors to Thiobacillus intermedius, T. perometabolis, T. neapolitanus, or T. acidophilus recipients, although they could be mated into these strains from T. novellus. All Thiobacillus species tested could transfer plasmids back to E. coli, with the exception of T. acidophilus. The donor-specific bacteriophages PRR1 and PRD1 were incapable of initiating the lytic cycle in RP4-bearing strains of T. novellus. The cosmid cloning vehicle pVK100 could be mobilized from E. coli to T. novellus with the aid of the "helper" plasmid pRK2013. pVK100 is stable in T. novellus, but pRK2013 is not maintained in this species. pRK2013 was also used to mobilize another cloning vector, R300B, to T. novellus. A previously unreported cryptic plasmid of approximately 24 megadaltons was observed in T. intermedius. No native plasmids were demonstrated in the other Thiobacillus species except in T. acidophilus, which contained cryptic plasmids ranging in size from 7.6 to 56 megadaltons (molecular mass).

Journal Article↗

Sulfur production by obligately chemolithoautotrophic thiobacillus species.

Transient-state experiments with the obligately autotrophic Thiobacillus sp. strain W5 revealed that sulfide oxidation proceeds in two physiological phases, (i) the sulfate-producing phase and (ii) the sulfur- and sulfate-producing phase, after which sulfide toxicity occurs. Specific sulfur-producing characteristics were independent of the growth rate. Sulfur formation was shown to occur when the maximum oxidative capacity of the culture was approached. In order to be able to oxidize increasing amounts of sulfide, the organism has to convert part of the sulfide to sulfur (HS(sup-)(symbl)S(sup0) + H(sup+) + 2e(sup-)) instead of sulfate (HS(sup-) + 4H(inf2)O(symbl)SO(inf4)(sup2-) + 9 H(sup+) + 8e(sup-)), thereby keeping the electron flux constant. Measurements of the in vivo degree of reduction of the cytochrome pool as a function of increasing sulfide supply suggested a redox-related down-regulation of the sulfur oxidation rate. Comparison of the sulfur-producing properties of Thiobacillus sp. strain W5 and Thiobacillus neapolitanus showed that the former has twice the maximum specific sulfide-oxidizing capacity of the latter (3.6 versus 1.9 (mu)mol/mg of protein/min). Their maximum specific oxygen uptake rates were very similar. Significant mechanistic differences in sulfur production between the high-sulfur-producing Thiobacillus sp. strain W5 and the moderate-sulfur-producing species T. neapolitanus were not observed. The limited sulfide-oxidizing capacity of T. neapolitanus appears to be the reason that it can convert only 50% of the incoming sulfide to elemental sulfur.

Journal Article↗

Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans.

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations at pH 4.0 revealed the presence of two distinguishable redox species with an E(m) of 510 mV and an E(m) of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of two low-spin hemes with a g(z)() of 3.08 (510 mV species) and a g(z)() of 3.22 (430 mV species). Modifications of the physicochemical properties of the cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al. (1996) Eur. J. Biochem. 242, 308-314], the gene of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.

Amino Acid Sequence↗

Thiobacillus sp. W5, the dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes.

The floating filter technique was successfully adapted for the isolation of the dominant, chemolithoautotrophic, sulfide-oxidizing bacterium from a sulfur-producing reactor after conventional isolation techniques had failed. The inoculated polycarbonate filters, floating on mineral medium, were incubated under gaseous hydrogen sulfide at non-toxic levels. This technique gave 200-fold higher recoveries than conventional isolation techniques. Viable counts on the filters, making up 15% of the total count, appeared to be all of the same species. Chemostat cultures of the new isolate had a very high sulfur-forming capacity, converting almost all hydrogen sulfide in the medium to elemental sulfur under high sulfide loads (27.5 mmol l-1 h-1) and fully aerobic conditions. This behaviour closely resembled that of the microbial community in the sulfur-producing reactor. Moreover, similar protein patterns were obtained by electrophoresis of cell-free extracts from the isolate and the mixed culture. It has therefore been concluded that this isolate represents the dominant sulfide-oxidizing population in the reactor. The isolate has been shown to be a new Thiobacillus species, related to Thiobacillus neapolitanus. In view of the general confusion currently surrounding the taxonomy of the thiobacilli, a new species has not been formally created. Instead, the isolate has been given the working name Thiobacillus sp. W5.

Aerobiosis↗