Thiobacillus Intermedius Research Articles

The cbbQ Genes, Located Downstream of the Form I and Form II RubisCO Genes, Affect the Activity of Both RubisCOs

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1 and cbbLS-2) and one form II type (cbbM) enzymes. The cbbQ-m gene was located downstream of cbbM, and showed high similarity to other cbbQ genes and nirQ/norQ genes in denitrifying bacteria. Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv. marinus closely resembled CbbQ from Thiobacillus intermedius and Thiobacillus neapolitannus and less closely resembled NirQ and NorQ. The cbbQ-m gene has been shown to activate the form II RubisCO in E. coli cells, and the cbbQ-t from Hydrogenophilus thermoluteolus could also activate the form II RubisCO. Both cbbQ genes have also been shown to activate the form I RubisCO from Hp. thermoluteolus in E. coli cells. However, the activation levels of two form I RubisCOs from Hv. marinus were smaller than that of form I RubisCOs from Hp. thermoluteolus. Form II RubisCO activated by CbbQ-m (QM) was purified from E. coli cells. The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ.

Toxicity of Cadmium Particle Dust in Bacterial Cells.

When Thiobacillus intermedius 13-1, Escherichia coli JM109, and Agrobacterium radiobacter IFO12665b1 were cultured in LB liquid medium containing 0.03 g or 0.1 g of cadmium particle dust, growth was strongly inhibited. Expose of cells to cadmium particle resulted in an increase in pH and concentration of ammonium ions. No cadmium particle attachment to the outer wall of T. intermedius or cell wall disruption was observed by transmission electron microscopy.

Effect of hypergravitational stress on microbial cell viability

Cell of Escherichia coli B, Thiobacillus intermedius 13-1, Bacillus amyloliquefaciens IFO14141, Staphylococcus aureus IID975, and Saccharomyces cerevisiae Kyokai no. 7 cultivated in nutrient media were subjected to hypergravitational stress for a period of between 1 and 24 h at 450,000 × g. The E. coli, B. amyloliquefaciens and S. cerevisiae cells showed survival rates of 38.5%, 0.005%, and 14.7%, respectively, after gravity treatment for 24 h as determined by their ability for colony formation, whereas a survival rate of S. cerevisiae cells of almost 100% was observed as determined using the methylene blue reduction test. E. coli cells, either in the logarithmic growth phase or cultivated in minimum media, were more sensitive to gravitational stress than those either in the stationary phase or cultivated in nutrient media.

Heavy metal particle resistance in Thiobacillus intermedius 13-1 isolated from corroded concrete

The effect of exposure to heavy metal particles on the growth and survival of bacterial cells was investigated. Thiobacillus intermedius 13-1, Escherichia coli JM109, and Agrobacterium radiobacter IFO12665b1 were cultured on LB solid medium or in 5 ml of liquid medium containing 0.03 or 0.1 g respectively of the heavy metals, aluminum, cadmium, iron, lead, molybdenum, nickel, and zinc. Cadmium, nickel and zinc strongly inhibited cell growth in the three strains. In contrast, the bacterial cells were not inhibited by aluminum, iron, or lead in the solid or liquid medium. When these bacteria were exposed to heavy metals by vigorous shaking for 10 min, lead and molybdenum, but not nickel and zinc, were markedly toxic to the bacterial cells. Different reaction were thus observed under low-level, long-term and high-level, short-term exposure conditions. T. intermedius showed more resistance to zinc and nickel, and was more sensitive to molybdenum exposure, than E. coli and A. radiobacter.

Effects of 60Co Gamma-Rays, Ultraviolet Light, and Mitomycin C on Halobacterium salinarium and Thiobacillus intermedius.

Lethal effects of 60Co gamma-rays, UV light, and mitomycin C on two kinds of bacteria, Halobacterium salinarium which grows in highly concentrated salt media and Thiobacillus intermedius which requires reduced sulfur compounds, were studied and compared with those on Escherichia coli B/r. D37 values for H. salinarium, T. intermedius and E. coli B/r were 393, 150, and 92 Gy, respectively, by exposure to 60Co gamma-rays. They were 212, 38, and 10 J/m2, respectively, by exposure to UV light and 2.36, 0.25, and 0.53 microgram/ml/h, respectively, by exposure to mitomycin C. Against these agents, H. salinarium was much more resistant than T. intermedius and E. coli B/r.

Use of Electroporation To Generate a Thiobacillus neapolitanus Carboxysome Mutant

Two cloning vectors designed for use in Escherichia coli and the thiobacilli were constructed by combining a Thiobacillus intermedius plasmid replicon with a multicloning site, lacZ(prm1), and either a kanamycin or a streptomycin resistance gene. Conditions necessary for the introduction of DNA into T. intermedius and T. neapolitanus via electroporation were examined and optimized. By using optimal electroporation conditions, the gene encoding a carboxysome shell protein, csoS1A, was insertionally inactivated in T. neapolitanus. The mutant showed a reduced number of carboxysomes and an increased level of CO(inf2) necessary for growth.

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.

Thiosulfate and tetrathionate degradation as well as biofilm generation by Thiobacillus intermedius and Thiobacillus versutus studied by microcalorimetry, HPLC, and ion-pair chromatography

Thiosulfate and tetrathionate degradation as well as biofilm generation by Thiobacillus intermedius and Thiobacillus versutus studied by microcalorimetry, HPLC, and ion-pair chromatography

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.

Cloning and expression of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli.

Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides, respectively. However, only the previously reported form I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A SalI fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5 alpha. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus. Other Calvin cycle genes were not clustered with either the form I or form II genes.

Cloning and expression of the ?-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Cloning and expression of the ?-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Characterization and identification of bacterial strains isolated from corroded concrete in the accumulation stratum and their resistance levels to heavy metals

Twenty thiosulfate-utilizing bacteria that may participate in the corrosion of concrete fabrications, were isolated from the accumulation stratum in the delta area of Hiroshima. Five of the isolated strains were selected for characterization, and four out of the five were classified into the genus Thiobacillus. Three of the five strains were further identified as Thiobacillus intermedius. The growth of these five strains in LB medium containing heavy metals was investigated. They showed strong resistance to cadmium and zinc ions.

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

The evolutionary relationship of the RuBisCO large subunit gene(s) (rbcL) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum, and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon, and Mastigocladus laminosus. In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus, and Thiothrix nivea. The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus, and T. nivea. The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus. It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

The evolutionary relationship of the RuBisCO large subunit gene(s) (rbcL) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum, and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon, and Mastigocladus laminosus. In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus, and Thiothrix nivea. The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus, and T. nivea. The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus. It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].

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.

Physiological Characteristics of the Facultatively Chemolithotrophic Thiobacillus Species Thiobacillus delicatus nom. rev., emend., Thiobacillus perometabolis, and Thiobacillus intermedius

The physiological characteristics of three facultatively chemolithotrophic Thiobacillus species with ubiquinone 8 (group II) (Thiobacillus delicatus THI 091T [T = type strain] Thiobacillus perometabolis THI 023 , and Thiobacillus intermedius THI 101T) were compared. These organisms differ from other facultatively chemolithotrophic Thiobacillus strains with ubiquinone 10 (group I) in requiring thiosulfate or elemental sulfur for optimum growth. T. delicatus THI 091T, T. perometabolis THI 023 , and T. intermedius THI 101T are regarded as three distinct species on the basis of their physiological characteristics. T. delicatus THI 091T differs from T. perometabolis THI 023T and T. intermedius THI 101T in its lack of motility, its ability to grow anaerobically with nitrate, and its intermediate formation of polythionate during thiosulfate oxidation. T. perometabolis THI 023T is distinguished from T. intermedius THI 101T by its ability to grow heterotrophically with a single carbon source. T. delicatus is not on the Approved Lists of Bacterial Names; therefore, this name is revived. In addition, the description of this organism is emended, considering its inability to grow heterotrophically with a single carbon source.

Ubiquinone, Fatty acid and DNA Base Composition Determination as a Guide to the Taxonomy of the Genus Thiobacillus

Facultatively chemolithotrophic thiobacilli. Thiobacillus perometabolis thi 022, Thiobacillus sp. A2, Thiobacillus novellus, Thiobacillus organoparus and Thiobacillus acidophilus, had ubiquinones with 10 isoprene units (Q-10) (group I). These species also had [octadecenoic acid (18:1) + cyclopropane acid of C19 (19cyc)] as major non-hydroxylated fatty acids. The organisms, except T. novellus, contained 3-hydroxydecanoic acid (3-OH 10:0) or 3-hydroxytetradecanoic acid (3-OH 14:0) as hydroxy fatty acids. DNA base compositions of these species were 63 to 68 mol% G + C. Other facultatively chemolithotrophic thiobacilli, including T. perometabolis thi 023, Thiobacillus delicatus and Thiobacillus intermedius contained Q-8 (group II). They had hexadecanoic acid (16:0), [hexadecenoic acid (16:1) + cyclopropane acid of C17 (17cyc)] and [18:1 + 19cyc] as major non-hydroxylated fatty acids. The species of group II had 3-OH 10:0 and some possessed 3-hydroxydodecanoic acid (3-OH 12:0). The DNA base composition of these strains was about 65 mol% G + C. Obligate chemolithotrophic thiobacilli contained Q-8 (group III). They had 16:0, [16:1 + 17cyc] with or without [18:1 + 19cyc] as major non-hydroxylated fatty acids. The species of group III contained 3-OH 12:0 and some had 3-OH 10:0 when grown at neutral pH and 3-OH 14:0 in acidophilic conditions. DNA base compositions of these organisms ranged from 51 to 64 mol% G + C. Chemotaxonomic characteristics would seem to be useful for the identification and the classification of thiobacilli.

Salinity requirements of a marine Thiobacillus intermedius

Thiobacillus intermedius was isolated from a salt marsh sediment with an interstitial water salinity of 30‰. This bacterium was cultured in a chemostat for 9 months. The optimum salinity for CO2 fixation by this Thiobacillus was 10‰, much less than the salinity of its natural environment. Respiration of cultures increased at high salinities and the pathway of thiosulfate oxidation was altered so that polythionates accumulated rapidly. One ecological conclusion from these results is that in nature this bacterium probably grows at its maximum possible rate only rarely.

Salinity requirements of a marine Thiobacillus intermedius: Smith, D.W. and S.F. Finazzo, 1981. Archs Microbiol., 129(3): 199–203

Salinity requirements of a marine Thiobacillus intermedius: Smith, D.W. and S.F. Finazzo, 1981. Archs Microbiol., 129(3): 199–203

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.