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1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, Moscow, 117312 Russia
2 G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pr. Nauki 5, Pushchino, Moscow Region, 142290 Russia
Correspondence
Grigorii I. Karavaiko
gregor{at}inmi.host.ru
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ABSTRACT |
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), there are three recognized species in the genus with validly published names: Sulfobacillus thermosulfidooxidans (type strain AT-1T=VKM B-1269T=DSM 9293T) (Golovacheva & Karavaiko, 1978), Sulfobacillus acidophilus (type strain NALT=DSM 10332T) (Norris et al., 1996) and Sulfobacillus sibiricus (type strain N1T=VKM B-2280T=DSM 17363T) (Melamud et al., 2003). Several unclassified Sulfobacillus strains have been described, e.g. strain L15 and strain RIV14 (Yahya et al., 1999) and strain Y0017 (Johnson et al., 2003), as well as uncultured clones from different environments (Ghauri et al., 2003; Okibe et al., 2003; Johnson et al., 2005). During pilot-scale tests of oxidation of a gold-containing sulfide concentrate by sulfobacilli and acidithiobacilli, aboriginal strain Kr1T was isolated. The profile exhibited for the restriction of chromosomal DNA was different from those of S. thermosulfidooxidans VKM B-1269T and S. sibiricus N1T, as revealed by pulsed-field gel electrophoresis (Kondrat'eva et al., 2003). In this paper, we report the characterization of strain Kr1T as the type strain of a novel species of Sulfobacillus.
An enrichment culture of strain Kr1T was initiated by inoculating the pulp of a gold-containing sulfide concentrate (10 %, v/v) into a modified (Melamud & Pivovarova, 1998) version of medium 9K, containing the following (g l–1): FeSO4.7H2O, 9.82; yeast extract, 0.2; (NH4)2SO4, 3.0; KCl, 0.1, K2HPO4, 0.5; MgSO4.7H2O, 0.5; Ca(NO3)2, 0.01; pH 2.0 (adjusted with 5 M H2SO4). The culture was incubated in 250 ml Erlenmeyer flasks with 100 ml of the aforementioned medium at 40 °C for 3 days on a rotary shaker at 180 r.p.m. A pure culture of strain Kr1T was obtained from the enrichment culture by means of serial decimal dilutions. Strain Kr1T could not grow autotrophically after two to three passages or organotrophically after three to four passages, nor could it grow on solid agar media or on a medium containing large amounts of organic substances. The purity of the culture of strain Kr1T was judged from the chromosomal DNA restriction profile, which remained unchanged throughout the experiments. Strain Kr1T was maintained in the modified 9K medium supplemented with 1 mM Na2S2O3 and passaged twice a month; the inoculum was added at increments of 10 % (v/v) to a final cell content of 107 ml–1.
The main phenotypic characteristics of strain Kr1T are summarized in Table 1. As revealed by light and electron microscopy performed as described previously (Melamud et al., 2003; Reynolds, 1963), vegetative cells of strain Kr1T were straight to slightly curved rods, 1.5–4.5 µm in length and 0.8–1.2 µm in diameter, being larger than the cells of other sulfobacilli (Table 1). Cells of strain Kr1T occurred singly or in chains of two to four cells; they lacked flagella. The cell wall, as viewed in ultrathin sections, was typical of Gram-positive bacteria; the S-layer was absent. Strain Kr1T produced spherical and oval, refractile endospores in subterminally swollen sporangia.
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). Therefore, strain Kr1T was thermotolerant and differed from the known moderately thermophilic sulfobacilli by having a lower temperature for growth (Table 1
). The pH range for growth in medium containing ferrous iron was pH 1.2–2.4, with an optimum at pH 2.0 (Fig. 1b). If grown on a medium containing S0, the pH range was 2.0–5.0, with an optimum at pH 2.5. Strain Kr1T and the other known sulfobacilli grew mixotrophically and oxidized mineral substrates (S0, ferrous iron and sulfide minerals) as energy sources in the presence of 0.02 % yeast extract or other organic compounds (Table 1). Of all the sulfobacilli, only strain N1T is unable to oxidize tetrathionate (Table 1); only S. acidophilus NALT was able to grow autotrophically on medium containing S0 in the presence of 5 % CO2 (Norris et al., 1996).
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and Owen et al. (1969), was 48.2±0.5 mol%, which is close to that of S. sibiricus N1T (48.2±0.2 mol%). S. thermosulfidooxidans VKM B-1269T and S. acidophilus NALT had DNA G+C contents of 47.5±0.2 and 56.0±1 mol%, respectively (Table 1). DNA–DNA hybridization was performed by using the optical reassociation method (De Ley et al., 1970). The data demonstrated a low level of interspecies relatedness: 11 % between strain Kr1T and S. acidophilus NALT, 33 % with S. thermosulfidooxidans VKM B-1269T and 44 % with S. sibiricus N1T. The level of DNA–DNA hybridization of strain Kr1T with itself was 100 %. The phylogenetic tree for the bacteria studied was constructed by using algorithms implemented in TREECON (Van de Peer & De Wachter, 1994). The 16S rRNA gene was amplified and sequenced as described by Edwards et al. (1989). A phylogenetic analysis showed that the 16S rRNA gene sequence of strain Kr1T fell within the phylogenetic cluster of species belonging to the genus Sulfobacillus (Fig. 2), the level of similarity being within the range 90.6–99.6 %. Therefore, strain Kr1T could not be related to any of the Sulfobacillus species with validly published names. Strain Kr1T formed a single cluster (100 % bootstrap support) showing a high degree of sequence similarity (98.3–99.6 %) with Sulfobacillus strains L15 and RIV14 (Yahya et al., 1999) and also with Sulfobacillus sp. Y0017 (Johnson et al., 2003) and uncultured Sulfobacillus sp. P3-5 from a pyrite-oxidizing bacterial population (GenBank accession no. AF460985). In addition, the isoprenoid quinone in strain Kr1T, as determined by the method of Collins (1985), was a menaquinone with seven isoprene units (MK-7). The presence of this menaquinone supported the affiliation of strains Kr1T to the family Alicyclobacillaceae, to which the genus Sulfobacillus belongs.
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Description of Sulfobacillus thermotolerans sp. nov.
Sulfobacillus thermotolerans (ther.mo.tol'er.ans. Gr. adj. thermos hot; L. part. adj. tolerans tolerating; N.L. part. adj. thermotolerans tolerating heat).
Cells are non-motile, aerobic, Gram-positive, endospore-forming rods, often occurring in chains composed of two to four cells. Rods are either straight or slightly curved, 1.5–4.5 µm in length and 0.8–1.2 µm in diameter. Oval and spherical endospores are located subterminally. Mixotrophic, oxidizing S0, ferrous iron, sulfide minerals and tetrathionate in the presence of 0.02 % yeast extract or other organic compounds (Table 1). Organotrophic growth is supported by the substrates indicated in Table 1. Autotrophic or organotrophic growth occurs only for a few passages. Thermotolerant: the temperature range is 20–60 °C and the optimum is 40 °C. Acidophilic: the pH range for growth is 1.2–2.4 and the optimum is pH 2.0. The DNA G+C content 48.2±0.5 mol%. The main isoprenoid quinone is MK-7.
The type strain, Kr1T (=VKM B-2339T=DSM 17362T), was isolated from sulfide ores.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Edwards, U., Rogall, T., Blocker, H., Emde, M. D. & Böttger, E. C. (1989). Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17, 7843–7853.
Ghauri, M. A., Khalid, A. M., Grant, S., Heaphy, S. & Grant, W. D. (2003). Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers. Extremophiles 7, 341–345.[Medline]
Golovacheva, R. S. & Karavaiko, G. I. (1978). Sulfobacillus, a new genus of thermophilic sporulating bacteria. Mikrobiologiia 47, 815–822 (in Russian).[Medline]
Johnson, D. B., Okibe, N. & Roberto, F. F. (2003). Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics. Arch Microbiol 180, 60–68.[CrossRef][Medline]
Johnson, D. B., Okibe, N. & Hallberg, K. B. (2005). Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis. J Microbiol Methods 60, 299–313.[CrossRef][Medline]
Karavaiko, G. I., Bogdanova, T. I., Tourova, T. P., Kondrat'eva, T. F., Tsaplina, I. A., Egorova, M. A., Krasil'nikova, E. N. & Zakharchuk, L. M. (2005). Reclassification of ‘Sulfobacillus thermosulfidooxidans subsp. thermotolerans’ strain K1 as Alicyclobacillus tolerans sp. nov. and Sulfobacillus disulfidooxidans Dufresne et al. 1996 as Alicyclobacillus disulfidooxidans comb. nov., and emended description of the genus Alicyclobacillus. Int J Syst Evol Microbiol 55, 941–947.
Kondrat'eva, T. F., Pivovarova, T. A., Muntyan, L. N., Ageeva, S. N. & Karavaiko, G. I. (2003). The strain genotypic heterogeneity of chemolitotrophic microorganisms. In 15th International Biohydrometallurgy Symposium. Proceedings of the International Symposium on Biohydrometallurgy: a Sustainable Technology in Evolution, pp. 1379–1389. Athens: Convention Centre of National Bank of Greece.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Melamud, V. S. & Pivovarova, T. A. (1998). Specific features of the growth of the type strain of Sulfobacillus thermosulfidooxidans in the 9K medium. Prikl Biokhim Mikrobiol 34, 309–315 (in Russian).
Melamud, V. S., Pivovarova, T. A., Tourova, T. P., Kalganova, T. V., Osipov, G. A., Lysenko, A. M., Kondrat'eva, T. F. & Karavaiko, G. I. (2003). Sulfobacillus sibiricus sp. nov., a novel moderately thermophilic bacterium. Mikrobiologiia 72, 681–688 (in Russian).[Medline]
Norris, P. A., Clark, D. A., Owen, J. P. & Waterhouse, S. (1996). Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria. Microbiology 142, 775–783.[Abstract]
Okibe, N., Gericke, M., Hallberg, K. B. & Johnson, D. B. (2003). Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation. Appl Environ Microbiol 69, 1936–1943.
Owen, R. J., Hill, L. R. & Lapage, S. P. (1969). Determination of DNA base composition from melting profiles in dilute buffers. Biopolymers 7, 503–516.[CrossRef][Medline]
Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17, 208–212.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows, a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Yahya, A., Roberto, F. F. & Johnson, D. B. (1999). Novel mineral-oxidizing bacteria from Montserrat (W.I.): physiological and phylogenetic characteristics. In Biohydrometallurgy and the Environment: Toward the Mining of the 21st Century, Process Metallurgy 9A, pp. 729–740. Edited by R. Amils & A. Ballester. Amsterdam: Elsevier.
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