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1 Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, Tampere, Finland
2 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
Correspondence
Anna H. Kaksonen
anna.kaksonen{at}tut.fi
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RL50JIIIT is DQ208688.
A phase-contrast micrograph of cells of strain RL50JIIIT is available as supplementary material in IJSEM Online.
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). The genus Desulfotomaculum was first described by Campbell & Postgate (1965). At the time of writing, the genus comprises 15 thermophilic species: D. alkaliphilum (Pikuta et al., 2000), D. arcticum (Vandieken et al., 2006), D. australicum (Love et al., 1993), D. carboxydivorans (Parshina et al., 2005), D. geothermicum (Daumas et al., 1988), D. kuznetsovii (Nazina et al., 1989), D. luciae (Liu et al., 1997), D. nigrificans (Werkman & Weaver, 1927; Campbell & Postgate, 1965), D. putei (Liu et al., 1997), ‘D. salinum’ (Nazina et al., 2005), D. solfataricum (Goorissen et al., 2003), D. thermoacetoxidans (Min & Zinder, 1990), D. thermobenzoicum with two subspecies, namely subsp. thermobenzoicum and subsp. thermosyntrophicum (Tasaki et al., 1991; Plugge et al., 2002), D. thermocisternum (Nilsen et al., 1996) and D. thermosapovorans (Fardeau et al., 1995). Recently, we reported the enrichment and isolation of thermophilic sulfate-reducers from an underground mine in a geothermally active area in Japan (Kaksonen et al., 2006). This paper describes a novel thermophilic, sulfate-reducing bacterium, strain RL50JIIIT, isolated from the mine. Strain RL50JIIIT was isolated from a geothermal mine site 250 m below ground, from a black sediment layer beneath a thin red layer of ferric iron on a tunnel wall. Temperature at the site was 70–80 °C. For comparisons, reference of D. kuznetsovii DSM 6115T, D. luciae DSM 12396T and D. solfataricum DSM 14956T, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
Enrichment and isolation of strain RL50JIIIT were performed at 50 °C using modified Postgate growth medium (pH 7.0–7.5) (Kaksonen et al., 2006) with lactate as the electron donor. Anaerobic roll-tubes solidified with 1.5 % agar were used for the isolation. For chemotaxonomic analysis and DNA isolation, the strain was cultured at 60 °C in modified DSM medium 641 containing lactate as the electron donor. The medium was supplemented with 1 ml selenate-tungstate solution l–1 (DSM medium 385) and sodium dithionate (25 mg l–1) was used as the reducing agent instead of Na2S.
The isolate was examined by phase-contrast microscopy (Axioskop 2; Zeiss) and photomicrographs were obtained using the agar slide technique as described by Kaksonen et al. (2004). Flagellum staining was performed as described by Heimbrook et al. (1989). Spore formation by the strain was examined microscopically and also by testing for growth after heat treatment (95 °C for 25 min). The Gram type of the cells was determined by both Gram staining and the KOH test (Gregersen, 1978).
The effects of temperature, pH and NaCl concentration on growth were determined as previously described (Kaksonen et al., 2006). The ability of the strain to utilize various electron donors (1–20 mM) was tested in medium containing 20 mM sulfate. The utilization of various electron acceptors (10 mM) was studied using lactate (10 mM) as electron donor. Amorphous iron(III) oxyhydroxide was formed by neutralizing FeCl3 solution to a pH of 7 with NaOH. The cultures were incubated for 1–2 weeks. Electron donor utilization was determined as bacterial growth (optical density at 660 nm, Shimadzu UV-1601 spectrophotometer, or Ultrospec II LKB Biochrom 4050 UV/visible spectrophotometer), hydrogen sulfide production or substrate conversion. Hydrogen sulfide production was determined spectrophotometrically and substrate conversion by GC as previously described (Kaksonen et al., 2004). Ferrous iron was determined colorimetrically (UV-1601; Shimadzu) with ferrozine (Stookey, 1970). Concentrations of sulfate, sulfite, thiosulfate, nitrate and nitrite were determined by ion chromatography (DX-120; Dionex).
Cell wall preparations were obtained by boiling cells in 20 % (w/v) aqueous trichloroacetic acid solution for 20 min. Diaminopimelic acid isomers were detected in cell-wall hydrolysates (4 M HCl, 100 °C, 16 h) by TLC on cellulose sheets (Merck) using the solvent system of Rhuland et al. (1955). Respiratory isoprenoid quinones were extracted and analysed according to the methods described by Collins & Jones (1981), Monciardini et al. (2003) and Groth et al. (1996) using an HPLC apparatus (Shimadzu) fitted with a reversed-phase C18 column [150 mm x 4.6 mm (ID), 5 µm, porosity 90 Å; Vydac] with UV detection at 269 nm. The identity of the quinones was verified by GC-MS (GCMS-QP2000; Shimadzu) using direct injection with a temperature increase of 60–250 °C at 35 °C min–1.
Fatty acid methyl esters of cellular fatty acids were obtained by saponification, methylation, extraction and base wash, as described by Kämpfer & Kroppenstedt (1996), Kroppenstedt (1985) and Miller (1982). The fatty acid methyl mixtures were separated by use of a gas chromatograph (5890 Series II; Hewlett Packard) equipped with a Hewlett Packard Ultra2 (cross-linked 5 % PH ME Siloxane) capillary column (25 mx0.2 mmx0.33 µm film thickness; HP Part No. 19091B - 102).
Methods for the amplification, sequencing and phylogenetic analysis of 16S rRNA genes were as described previously (Kaksonen et al., 2006). Genomic DNA for G+C content determination and DNA–DNA hybridization experiments was released by rupturing cells using a French pressure cell (Thermo Spectronic) and then purified by chromatography on hydroxyapatite (Cashion et al., 1977). DNA was hydrolysed with P1 nuclease and the nucleotides dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989). The G+C content of the resulting deoxyribonucleosides was determined by reversed-phase HPLC (Shimadzu) and calculated from the ratio of deoxyguanosine (dG) and thymidine (dT) (Tamaoka & Komagata, 1984; Mesbah et al., 1989). DNA–DNA hybridization experiments were performed at DSMZ according to the method of De Ley et al. (1970) with the modifications described by Huß et al. (1983), Escara & Hutton (1980) and Jahnke (1992) using a spectrophotometer (model 2600; Gilford) equipped with a thermoprogrammer and plotter (model 2527-R; Gilford).
Cells of strain RL50JIIIT were straight or slightly curved rods, 0.8–1.0 µm in diameter and 3–10 µm in length (see Supplementary Fig. S1 in IJSEM Online). The strain formed spherical spores, which were located centrally or subterminally. The spores were able to germinate after a heat shock at 95 °C for 25 min. Sporulation caused swelling of the cells, giving them a lemon-shaped appearance. The cells were motile with two or more flagella and Gram-positive as determined by both Gram-staining and the KOH test. The temperature, pH and NaCl ranges for growth of strain RL50JIIIT are given in Table 1. The temperature at the sampling point was 70–80 °C, which is at or above the upper temperature limit of growth of strain RL50JIIIT. It is possible that the strain was present in the habitat as spores. Strain RL50JIIIT was able to use sulfate, sulfite, thiosulfate and elemental sulfur as electron acceptors. Electron donors utilized by the strain included H2 in the presence of CO2, alanine and various carboxylic acids, or their sodium salts, and alcohols (Table 1). A number of electron donors (e.g. propionate, butyrate, pentanoate, pyruvate, butanol, ethanol, lactate, isobutyrate, 2-methyl butyrate and succinate) were oxidized to acetate, whereas no acetate accumulated during the oxidation of others (e.g. alanine, hexadecanoate and nonanoate). The strain fermented lactate and pyruvate.
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. Significant proportions of saturated iso-branched fatty acids were found in strain RL50JIIIT, the major components being iso-C15 : 0, iso-C17 : 0 DMA (dimethyl acetal), iso-C15 : 0 DMA and iso-C17 : 0 (Table 2).
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). A phylogenetic tree constructed on the basis of 16S rRNA gene sequences showed that the closest relative of strain RL50JIIIT was D. solfataricum DSM 14956T (Fig. 1), with which it shared 98.7 % sequence similarity in the ARB similarity matrix (which omits insertions and deletions from the pairwise comparison). Strain RL50JIIIT shared a sequence similarity of 96.2 % with D. kuznetsovii DSM 6115T in the ARB similarity matrix and of 94.2 % with D. luciae DSM 12396T. The 16S rRNA gene sequence of strain RL50JIIIT has a 104-bp insert, which is not present in D. solfataricum or D. kuznetsovii, but which shows 95.2 % similarity to that in the 16S rRNA gene sequence of D. luciae. 16S rRNA gene sequence similarities of strain RL50JIIIT with the type strains of D. solfataricum, D. kuznetsovii and D. luciae as calculated with the BIOEDIT program (which includes insertions and deletions in the pairwise comparison) were 92, 90 and 94 %, respectively. Levels of DNA–DNA relatedness between strain RL50JIIIT and D. solfataricum DSM 14956T, D. kuznetsovii DSM 6115T and D. luciae DSM 12396T were 47.4, 59.5 and 47.8 %, respectively, which justifies a novel species designation for strain RL50JIIIT (Wayne et al., 1987).
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), the ability or inability to use certain electron acceptors and donors (Table 1), and a higher proportion of iso-C15 : 0 DMA and iso-C17 : 0 DMA and a lower proportion of C16 : 0 and iso-C17 : 0 in the whole-cell fatty acids (Table 2). Based on these phenotypic and genomic differences, strain RL50JIIIT can be assigned as the type strain of a novel species of the genus Desulfotomaculum, for which the name Desulfotomaculum thermosubterraneum sp. nov. is proposed.
Description of Desulfotomaculum thermosubterraneum sp. nov.
Desulfotomaculum thermosubterraneum (ther.mo.sub.ter.ra'ne.um. Gr. adj. thermos hot; L. adj. neut. subterraneum subterranean, underground, below the Earth's surface; thermosubterraneum thermophilic inhabitant of the Earth's subsurface).
Cells are motile, Gram-positive, spore-forming rods (0.8–1.0x3–10 µm). Growth occurs at 50–72 °C (optimum 61–66 °C), pH 6.4–7.8 (optimum pH 7.2–7.4) and NaCl concentration of 0–1.5 % (optimum 0–1 % NaCl). Sulfate, sulfite, thiosulfate and elemental sulfur are used as electron acceptors. The following substrates are used as electron donors in the presence of sulfate: H2 in the presence of CO2, alanine, various carboxylic acids and alcohols (see Table 1). Fermentative growth occurs on lactate and pyruvate. The cell wall contains mesodiaminopimelic acid as the diagnostic diamino acid. The major isoprenoid quinone is MK-7. Major whole-cell fatty acids are iso-C15 : 0, iso-C17 : 0 DMA, iso-C15 : 0 DMA and iso-C17 : 0. The G+C content of the DNA is 54.4 mol%.
The type strain, RL50JIIIT (=DSM 16057T=JCM 13837T), was isolated from a geothermally active underground mine in Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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