Sulfobacillus Thermotolerans Sp. Nov., a Thermotolerant, Chemolithotrophic Bacterium

International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1039–1042 DOI 10.1099/ijs.0.64106-0

Sulfobacillus thermotolerans sp. nov., a thermotolerant, chemolithotrophic bacterium

Tat’yana I. Bogdanova,1 Iraida A. Tsaplina,1 Tamara F. Kondrat’eva,1 Vitalii I. Duda,2 Natalya E. Suzina,2 Vitalii S. Melamud,1 Tat’yana P. Tourova1 and Grigorii I. Karavaiko1

Correspondence 1Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Grigorii I. Karavaiko Oktyabrya 7/2, Moscow, 117312 Russia [email protected] 2G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pr. Nauki 5, Pushchino, Moscow Region, 142290 Russia

A thermotolerant, Gram-positive, aerobic, endospore-forming, acidophilic bacterium (strain Kr1T) was isolated from the pulp of a gold-containing sulfide concentrate processed at 40 6Cina gold-recovery plant (Siberia). Cells of strain Kr1T were straight to slightly curved rods, 0?8–1?2 mm in diameter and 1?5–4?5 mm in length. Strain Kr1T formed spherical and oval, refractile, subterminally located endospores. The temperature range for growth was 20–60 6C, with an optimum at 40 6C. The pH range for growth on medium containing ferrous iron was 1?2–2?4, with an optimum at pH 2?0; the pH range for growth on medium containing S0 was 2?0–5?0, with an optimum at pH 2?5. Strain Kr1T was mixotrophic, oxidizing ferrous iron, S0, tetrathionate or sulfide minerals as energy sources in the presence of 0?02 % yeast extract or other organic substrates. The G+C content of the DNA of strain Kr1T was 48?2±0?5 mol%. Strain Kr1T showed a low level of DNA–DNA reassociation with the known Sulfobacillus species (11–44 %). 16S rRNA gene sequence analysis revealed that Kr1T formed a separate phylogenetic group with a high degree of similarity between the nucleotide sequences (98?3–99?6 %) and 100 % bootstrap support within the phylogenetic Sulfobacillus cluster. On the basis of its physiological properties and the results of phylogenetic analyses, strain Kr1T can be affiliated to a novel species of the genus Sulfobacillus, for which the name Sulfobacillus thermotolerans sp. nov. is proposed. The type strain is Kr1T (=VKM B-2339T=DSM 17362T).

The genus Sulfobacillus includes Gram-positive, endospore- sulfide concentrate by sulfobacilli and acidithiobacilli, forming, acidophilic bacteria that obtain energy by oxidiz- aboriginal strain Kr1T was isolated. The profile exhibited ing ferrous iron, elemental sulfur and sulfide minerals in for the restriction of chromosomal DNA was different the presence of 0?02 % yeast extract. To date, following from those of S. thermosulfidooxidans VKM B-1269T and S. the reclassification of Sulfobacillus disulfidooxidans in the sibiricus N1T, as revealed by pulsed-field gel electrophoresis genus Alicyclobacillus (Karavaiko et al., 2005), there are (Kondrat’eva et al., 2003). In this paper, we report the three recognized species in the genus with validly pub- characterization of strain Kr1T as the type strain of a novel lished names: Sulfobacillus thermosulfidooxidans (type species of Sulfobacillus. T= T= T strain AT-1 VKM B-1269 DSM 9293 ) (Golovacheva T & Karavaiko, 1978), Sulfobacillus acidophilus (type strain An enrichment culture of strain Kr1 was initiated by T T inoculating the pulp of a gold-containing sulfide con- NAL =DSM 10332 ) (Norris et al., 1996) and Sulfobacillus T T T centrate (10 %, v/v) into a modified (Melamud & sibiricus (type strain N1 =VKM B-2280 =DSM 17363 ) Pivovarova, 1998) version of medium 9K, containing the (Melamud et al., 2003). Several unclassified Sulfobacillus following (g l21): FeSO .7H O, 9?82; yeast extract, 0?2; strains have been described, e.g. strain L15 and strain RIV14 4 2 (NH ) SO ,3?0; KCl, 0?1, K HPO ,0?5; MgSO .7H O, (Yahya et al., 1999) and strain Y0017 (Johnson et al., 2003), 4 2 4 2 4 4 2 0?5; Ca(NO ) ,0?01; pH 2?0 (adjusted with 5 M H SO ). as well as uncultured clones from different environments 3 2 2 4 The culture was incubated in 250 ml Erlenmeyer flasks (Ghauri et al., 2003; Okibe et al., 2003; Johnson et al., 2005). with 100 ml of the aforementioned medium at 40 uC for During pilot-scale tests of oxidation of a gold-containing 3 days on a rotary shaker at 180 r.p.m. A pure culture of strain Kr1T was obtained from the enrichment culture T The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene by means of serial decimal dilutions. Strain Kr1 could sequence of strain Kr1T is DQ124681. not grow autotrophically after two to three passages or

64106 G 2006 IUMS Printed in Great Britain 1039 T. I. Bogdanova and others organotrophically after three to four passages, nor could it lower temperature for growth (Table 1). The pH range grow on solid agar media or on a medium containing for growth in medium containing ferrous iron was pH 1?2– large amounts of organic substances. The purity of the 2?4, with an optimum at pH 2?0 (Fig. 1b). If grown on culture of strain Kr1T was judged from the chromosomal a medium containing S0, the pH range was 2?0–5?0, with DNA restriction profile, which remained unchanged an optimum at pH 2?5. Strain Kr1T and the other known throughout the experiments. Strain Kr1T was maintained sulfobacilli grew mixotrophically and oxidized mineral in the modified 9K medium supplemented with 1 mM substrates (S0, ferrous iron and sulfide minerals) as energy Na2S2O3 and passaged twice a month; the inoculum was sources in the presence of 0?02 % yeast extract or other added at increments of 10 % (v/v) to a final cell content of organic compounds (Table 1). Of all the sulfobacilli, only 107 ml21. strain N1T is unable to oxidize tetrathionate (Table 1); only S. acidophilus NALT was able to grow autotrophically on T 0 The main phenotypic characteristics of strain Kr1 are medium containing S in the presence of 5 % CO2 (Norris summarized in Table 1. As revealed by light and electron et al., 1996). microscopy performed as described previously (Melamud et al., 2003; Reynolds, 1963), vegetative cells of strain Kr1T The G+C content of the genomic DNA of strain Kr1T, were straight to slightly curved rods, 1?5–4?5 mm in length determined by using the methods of Marmur (1961) and and 0?8–1?2 mm in diameter, being larger than the cells of Owen et al. (1969), was 48?2±0?5 mol%, which is close to other sulfobacilli (Table 1). Cells of strain Kr1T occurred that of S. sibiricus N1T (48?2±0?2 mol%). S. thermosulfido- T T singly or in chains of two to four cells; they lacked flagella. oxidans VKM B-1269 and S. acidophilus NAL had DNA The cell wall, as viewed in ultrathin sections, was typical G+C contents of 47?5±0?2 and 56?0±1 mol%, respec- of Gram-positive bacteria; the S-layer was absent. Strain tively (Table 1). DNA–DNA hybridization was performed Kr1T produced spherical and oval, refractile endospores in by using the optical reassociation method (De Ley et al., subterminally swollen sporangia. 1970). The data demonstrated a low level of interspecies relatedness: 11 % between strain Kr1T and S. acidophilus The temperature range for growth of strain Kr1T was NALT, 33 % with S. thermosulfidooxidans VKM B-1269T and 20–60 uC, with an optimum at 40 uC (Fig. 1a). Therefore, 44 % with S. sibiricus N1T. The level of DNA–DNA strain Kr1T was thermotolerant and differed from the hybridization of strain Kr1T with itself was 100 %. The known moderately thermophilic sulfobacilli by having a phylogenetic tree for the bacteria studied was constructed

Table 1. Characteristics of strains of Sulfobacilus species

Strains: 1, S. thermotolerans sp. nov. Kr1T;2,S. thermosulﬁdooxidans VKM B-1269T (data from Golovacheva & Karavaiko, 1978); 3, S. acidophilus NALT (Norris et al., 1996); 4, S. sibiricus N1T (Melamud et al., 2003). All cells were rod-shaped and spore-forming and grew on yeast extract.

Characteristic 1 2 3 4

Cell size (mm) 0?8–1?261?5–4?50?6–0?861–3?00?5–0?863?0–5?00?7–1?161?0–3?0 Growth pH range (optimum) 1?2–2?4(2?0) 1?5–5?5(1?7–2?4) (2?0) 1?1–2?6(2?0) Growth temperature range (optimum) (uC) 20–60 (40) 20–60 (50–55) (45–50) 17–60 (55) DNA G+C content (mol%) 48?2±0?547?2–47?556±148?2±0?2 2+ 0 2À 2+ 0 2À 2À 2+ 0 2+ 0 Mineral substrate(s) Fe ,S,S4O6 , Fe ,S,S2O3 ,S4O6 , Fe ,S, sulfide Fe ,S, sulfide minerals sulfide minerals minerals sulfide minerals Growth on: Malate + 222 Fructose +++2 Glucose +++2 Sucrose +++2 Glutathione (reduced) ++22 Casein 2 + 22 Trehalose 2 + 22 Mannose 2 + 22 Raffinose 2 + 22 Glutamate 2 + 22 Ribose 22+ 2 + + + + Minimum generation time (h) 2?0 (Fe2 );1?8(S0)2?5 (Fe2 ); 6 (S0)3?5 (Fe2 ); 6?8 1?4 (Fe2 ); 3?5(S0) (yeast extract)

1040 International Journal of Systematic and Evolutionary Microbiology 56 Sulfobacillus thermotolerans sp. nov.

Fig. 2. Phylogenetic tree showing the position of strain Kr1T among members of the genus Sulfobacillus of the phylum ‘Bacillus–Clostridium’ of the Gram-positive bacteria. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points; values greater than 95 % were consid- ered signiﬁcant. Bar, 2 nucleotide substitutions per 100 nucleo- tides (Jukes–Cantor distance).

Description of Sulfobacillus thermotolerans sp. nov.

Fig. 1. Dependence of the maximum speciﬁc growth rate (mmax) Sulfobacillus thermotolerans (ther.mo.tol9er.ans. Gr. adj. on temperature (a) and pH (b). thermos hot; L. part. adj. tolerans tolerating; N.L. part. adj. thermotolerans tolerating heat).

Cells are non-motile, aerobic, Gram-positive, endospore- by using algorithms implemented in TREECON (Van de Peer forming rods, often occurring in chains composed of two & De Wachter, 1994). The 16S rRNA gene was amplified and to four cells. Rods are either straight or slightly curved, sequenced as described by Edwards et al. (1989). A phylo- 1?5–4?5 mm in length and 0?8–1?2 mm in diameter. Oval genetic analysis showed that the 16S rRNA gene sequence of T and spherical endospores are located subterminally. Mixo- strain Kr1 fell within the phylogenetic cluster of species trophic, oxidizing S0, ferrous iron, sulfide minerals and belonging to the genus Sulfobacillus (Fig. 2), the level of tetrathionate in the presence of 0?02 % yeast extract or other similarity being within the range 90?6–99?6 %. Therefore, organic compounds (Table 1). Organotrophic growth is strain Kr1T could not be related to any of the Sulfobacillus T supported by the substrates indicated in Table 1. Autotro- species with validly published names. Strain Kr1 formed phic or organotrophic growth occurs only for a few passages. a single cluster (100 % bootstrap support) showing a high Thermotolerant: the temperature range is 20–60 uC and the degree of sequence similarity (98?3–99?6 %) with Sulfobac- optimum is 40 uC. Acidophilic: the pH range for growth is illus strains L15 and RIV14 (Yahya et al., 1999) and also with 1?2–2?4 and the optimum is pH 2?0. The DNA G+C Sulfobacillus sp. Y0017 (Johnson et al., 2003) and uncultured content 48?2±0?5 mol%. The main isoprenoid quinone is Sulfobacillus sp. P3-5 from a pyrite-oxidizing bacterial MK-7. population (GenBank accession no. AF460985). In addition, the isoprenoid quinone in strain Kr1T, as determined by the The type strain, Kr1T (=VKM B-2339T=DSM 17362T), method of Collins (1985), was a menaquinone with seven was isolated from sulfide ores. 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. Acknowledgements

Hence, on the basis of its physiological properties and the This work was supported by the following grants: ‘Leading Scientiﬁc T Schools’ N 1742.2003.4; Initiative Scientiﬁc Projects RFFI N 03-04- results of phylogenetic analyses, strain Kr1 represents a 49294 and 05-04-48058; and State Contract N 43.073.1.1.2515. The novel species of the genus Sulfobacillus, for which the name authors wish to express their gratitude to A. M. Lysenko for DNA Sulfobacillus thermotolerans sp. nov. is proposed. analysis and DNA–DNA hybridization. http://ijs.sgmjournals.org 1041 T. I. Bogdanova and others

References Biohydrometallurgy: a Sustainable Technology in Evolution, pp. 1379–1389. Athens: Convention Centre of National Bank of Collins, M. D. (1985). Analysis of isoprenoid quinones. Methods Greece. Microbiol 18, 323–363. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative acid from microorganisms. J Mol Biol 3, 208–218. measurement of DNA hybridization from renaturation rates. Eur Melamud, V. S. & Pivovarova, T. A. (1998). Specific features of the J Biochem 12, 133–142. growth of the type strain of Sulfobacillus thermosulfidooxidans in the Edwards, U., Rogall, T., Blocker, H., Emde, M. D. & Bo¨ ttger, E. C. 9K medium. Prikl Biokhim Mikrobiol 34, 309–315 (in Russian). (1989). Isolation and direct complete nucleotide determination of Melamud, V. S., Pivovarova, T. A., Tourova, T. P., Kalganova, T. V., entire genes. Characterization of a gene coding for 16S ribosomal Osipov, G. A., Lysenko, A. M., Kondrat’eva, T. F. & Karavaiko, G. I. RNA. Nucleic Acids Res 17, 7843–7853. (2003). Sulfobacillus sibiricus sp. nov., a novel moderately thermo- Ghauri, M. A., Khalid, A. M., Grant, S., Heaphy, S. & Grant, W. D. philic bacterium. Mikrobiologiia 72, 681–688 (in Russian). (2003). Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes Norris, P. A., Clark, D. A., Owen, J. P. & Waterhouse, S. (1996). using integron-specific primers. Extremophiles 7, 341–345. Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria. Microbiology Golovacheva, R. S. & Karavaiko, G. I. (1978). Sulfobacillus, a new 142, 775–783. genus of thermophilic sporulating bacteria. Mikrobiologiia 47, 815– 822 (in Russian). Okibe, N., Gericke, M., Hallberg, K. B. & Johnson, D. B. (2003). Enumeration and characterization of acidophilic microorganisms Johnson, D. B., Okibe, N. & Roberto, F. F. (2003). Novel thermo- isolated from a pilot plant stirred-tank bioleaching operation. Appl acidophilic bacteria isolated from geothermal sites in Yellowstone Environ Microbiol 69, 1936–1943. National Park: physiological and phylogenetic characteristics. Arch Microbiol 180, 60–68. Owen, R. J., Hill, L. R. & Lapage, S. P. (1969). Determination of DNA base composition from melting profiles in dilute buffers. Biopolymers Johnson, D. B., Okibe, N. & Hallberg, K. B. (2005). Differentiation 7, 503–516. and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction Reynolds, E. S. (1963). The use of lead citrate at high pH as enzyme analysis. J Microbiol Methods 60, 299–313. an electron-opaque stain in electron microscopy. J Cell Biol 17, Karavaiko, G. I., Bogdanova, T. I., Tourova, T. P., Kondrat’eva, T. F., 208–212. Tsaplina, I. A., Egorova, M. A., Krasil’nikova, E. N. & Zakharchuk, L. M. Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows, a (2005). Reclassification of ‘Sulfobacillus thermosulfidooxidans subsp. software package for the construction and drawing of evolutionary thermotolerans’ strain K1 as Alicyclobacillus tolerans sp. nov. and trees for the Microsoft Windows environment. Comput Appl Biosci Sulfobacillus disulfidooxidans Dufresne et al. 1996 as Alicyclobacillus 10, 569–570. disulfidooxidans comb. nov., and emended description of the genus Yahya, A., Roberto, F. F. & Johnson, D. B. (1999). Novel Alicyclobacillus. Int J Syst Evol Microbiol 55, 941–947. mineral-oxidizing bacteria from Montserrat (W.I.): physiological Kondrat’eva, T. F., Pivovarova, T. A., Muntyan, L. N., Ageeva, S. N. and phylogenetic characteristics. In Biohydrometallurgy and the & Karavaiko, G. I. (2003). The strain genotypic heterogeneity of Environment: Toward the Mining of the 21st Century, Process chemolitotrophic microorganisms. In 15th International Biohydro- Metallurgy 9A, pp. 729–740. Edited by R. Amils & A. Ballester. metallurgy Symposium. Proceedings of the International Symposium on Amsterdam: Elsevier.

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