Tatsuo Tano

Acidobacterium capsulatum gen. nov., sp. nov.: An acidophilic chemoorganotrophic bacterium containing menaquinone from acidic mineral environment

Acidobacterium is proposed as a new genus for the acidophilic, chemoorganotrophic bacteria containing menaquinone isolated from acidic mineral environments.Acidobacterium capsulatum is proposed for the singleAcidobacterium species which consists of eight strains (Biogroup 5). The members of this species are gram-negative, aerobic, mesophilic, non-spore-forming, capsulated, saccharolytic, and rod-shaped bacteria. They are motile by peritrichous flagella. They can grow between pH 3.0 and 6.0, but not at pH 6.5. They give positive results in tests for esculin hydrolysis, catalase, and β-galactosidase. Oxidase and urease are negative. They can use glucose, cellobiose, starch, maltose, or β-gentiobiose as a sole carbon source, but cannot use elemental sulfur and ferrous iron as an energy source. The DNA base composition is 59.7–60.8 guanine plus cytosine (G+C) mol%. The major isoprenoid quinone is menaquinone with eight isoprene units (MK-8). The major fatty acid is 13-methyltetradecanoic acid. DNA relatedness between this species and the species ofAcidiphilium, Acidomonas, andDeinobacter was 18 to 2%. From phenotypic and chemotaxonomic characters, these member do not belong to any known taxa of gram-negative bacteria. A culture of the type strain (strain 161) has been deposited in the Japan Collection of Microorganisms as JCM 7670.

Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the Genus Acidocella gen. nov., and Emendation of the Genus Acidiphilium

Summary Phylogenetic relationships among members of the genus Acidiphilium and related genera were elucidated by studying 16S rRNA gene sequence information. Approximately 1.5 kbp fragments of 16S rDNAs from the test organisms were amplified by the polymerase chain reaction and sequenced directly by the combined method consisting of cycle sequencing and automated fluorescence detection. Sequence comparisons and evolutionary distance analyses with the sequence from some reference species showed that all members of the genus Acidiphilium formed a cluster within the alpha- I subclass of the Proteobacteria, with Acidomonas methanolica, Acetobacter aceti, and Rhodopila globiformis as the nearest phylogenetic neighbors. The Acidiphilium cluster was divided into two subclusters whose monophyly was supported by high levels of bootstrap confidence: one included A. facilis and A. aminolytica, and the other consisted of all other Acidiphilium species. The levels of sequence similarity between species of the different subclusters were less than 94.2%. The A. facilis-A. aminolytica group is also distinguishable from the other group in a number of phenotypic and chemotaxonomic characteristics. Thus we propose to transfer A. aminolytica and A. facilis to a new genus, Acidocella gen. nov., with Acidocella facilis as the type species. Following this proposal, the description of the genus Acidiphilium was emended.

Actual substrate for elemental sulfur oxidation by sulfur:ferric ion oxidoreductase purified from Thiobacillus ferrooxidans

Initial step of elemental sulfur (S0) oxidation by a purified sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans was investigated. When S0 and reduced glutathione (GSH), whichwas absolutely required for S0 oxidation by sulfur:ferric ion oxidoreductase, were incubated in a buffer solution (pH 6.5), hydrogen sulfide (H 2S) and GSSG were chemically produced at the rate of 0.021 and 0.082 μmol/ml per h, respectively. If sulfur:ferric ion oxidoreductase was addedto the incubation mixture, H2S production immediately stopped and sulfite production opened, suggesting that H2S is an actual substrate of sulfu:ferric ion oxidoreductase. Amongthe reduced sulfur compounds tested, S0, H2S and FeS were utilized as an electron donorof sulfur:ferric ion oxidoreductase and a mechanism of initial steps of S0 oxidation was proposed. It was also found that when S0 was oxidized by sulfur:ferric ion oxidoreductase in the presence of GSH, contact of sulfur:ferric ion oxidoreductase with solid element sulfur was unnecessary.

Purification and Some Properties of a Tetrathionate Decomposing Enzyme from Thiobacillus thiooxidans.

A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104,000, and was composed of two identical subunits (MW = 58,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0-3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mm sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mm of sulfate ion was 2.4 mm. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mm. Though cupric ion up to 0.01 mm markedly stimulated the activity in the presence of 20 mm sulfate ion, a higher concentration (1 mm) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.

Acidiphilium aminolytica sp. nov.: An acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment

Acidiphilium aminolytica is proposed for a species of the genusAcidiphilium. Acidiphilium aminolytica can be phenotypically differentiated from all other species of the genusAcidiphilium. The seven strains of this species that have been studied are Gram-negative, aerobic, mesophilic, non-sporeforming, motile, and rod-shaped bacteria. They grow between pH 3.0 and 6.0, but not at pH 6.5. They yield positive results in tests for hippuric acid hydrolysis, catalase and urease production. Oxidase, esculin hydrolysis, and β-galactosidase tests are negative. They can used-glucose,d-galactose, inositol, sorbitol,l-lysine,l-glutamate,l-arginine, β-alanine,dl-4-aminobutyrate,dl-5-aminovalerate, sperimine, or diaminobutane as a sole carbon source, but cannot use elemental sulfur and ferrous iron as an energy source. The DNA base composition is 58.7–59.2 G+C mol%. The major isoprenoid quinone is ubiquinone with ten isoprene unit (Q-10). The major fatty acid is the C18:1 fatty acid. Two ornithine amide lipids, the C18:1 fatty acid esters of α-N-3-hydroxystearylornithyltaurine and α-N-3-hydroxystearylornithine, are detected as the polar aminolipid. DNA relatedness between this species and the other species ofAcidiphilium, the generaAcidomonas, andAcidobacterium was 29 to 2%. These results indicate, that this new species should be placed in the genusAcidiphilium. The type strain (strain 101) ofA. aminolytica is JCM 8796.

Purification and Properties of an Acidic β-Glucosidase from Acidobacterium capsulatum

Abstract Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.

Growth Inhibition of Acidiphilium Species by Organic Acids Contained in Yeast Extract

Abstract The growth of Acidiphilium species was inhibited by 0.4% yeast extract (Difco Laboratories). The growth inhibitory substances were purified from the yeast extract and identified as dl -lactic acid and succinic acid. The minimum inhibitory concentrations (MICs) of dl -lactic and succinic acids against Acidiphilium sp. 63 were 1 and 3 mM, respectively. The contents of dl -lactic and succinic acids in the yeast extracts were estimated at 43.9 and 1.2 mg/g using high-performance liquid chromatography. Acidiphilium species that we have been studying could be classified into two groups on the bases of the difference in susceptibility to these two organic acids.

Cloning and expression of the Thiobacillus ferrooxidans 3-isopropylmalate dehydrogenase gene in Escherichia coli

Abstract The 3-isopropylmalate (3-IPM) dehydrogenase [EC 1.1.1.85] gene leuB of an acidophilic autotroph Thiobacillus ferrooxidans was cloned and expressed in Escherichia coli . Recombinant plasmids pTFL1 and pTFL2, carrying a 6.7-kb Pst I fragment in the opposite orientation, conferred the same level of 3-IPM dehydrogenase activity on an E. coli leuB mutant. Restriction endonuclease mapping and deletion analysis indicated that 3-IPM dehydrogenase gene was on a 3.1-kb Pst I- Nru I fragment. The expression of the 3-IPM dehydrogenase gene of T. ferrooxidans was repressed by the addition of leucine in the culture medium of E. coli carrying the plasmid pTFL1. These results indicate that the 6.7-kb fragment contains the leu B structural gene and its regulatory region.

Growth of moderately thermophilic iron-oxidizing bacterium strain TI-1 in synthetic medium

Abstract The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO 2 fixation, grows at 45°C in Fe 2+ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10 8 cells per ml, but does not grow in Fe 2+ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe 2+ -6AA, containing Fe 2+ , salts, and the following six l -amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10 8 cells/ml. The six amino acids in the Fe 2+ -6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe 2+ (10 mM)-6AA medium supplemented with 21 mM Fe 3+ , reduction of Fe 3+ to Fe 2+ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe 3+ to Fe 2+ .

Restriction endonuclease AfaI from Acidiphilium facilis, a new isoschizomer of RsaI: purification and properties

Abstract We have purified Afa I endonuclease, an isoschizomer of Rsa I, from Acidiphilium facilis strain 28H. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis, and composed of a single polypeptide chain with a molecular weight of 30 000. Afa I endonuclease, like Rsa I, recognizes the tetranucleotide sequence 5′-G-T-A-C-3′, and cleaves between the T and A to produce blunt-ended fragments. The yield of the enzyme is 50–100-times that of the Rsa I, which is form a phototrophic bacterium, Rhodospseudomonas sphaeroides strain 28/5.