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Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake, Kyoto 606-8502, Japan
Correspondence
Reiji Tanaka
tanakar{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Hokai et al., 1995). Heterotrophic aerobes belonging to the genera Aeropyrum, Pyrobaculum, Thermus, Rhodothermus and Thermaerobacter (Sako et al., 1996a, b; Völkl et al., 1993; Oshima & Imahori, 1974; Alfredsson et al., 1979; Takai et al., 1999) as well as hydrogen-oxidizers belonging to the genera Aquifex and Hydrogenivirga (Huber et al., 1992; Nakagawa et al., 2004) have been isolated from hydrothermal beaches. These observations indicate that coastal hydrothermal environments with high oxygen concentrations harbour a highly diverse group of marine thermophiles; further exploration of these environments may lead to the discovery of novel thermophilic micro-organisms. Here, we describe the isolation of a novel thermophilic, strictly aerobic bacterium from a coastal hydrothermal beach in Japan.
Sandy sediments and hot fluids were collected from a hydrothermal beach on the Satsuma peninsula, Kagoshima, Japan. The temperature of the vent fluids was 90–95 °C. Samples were stored aerobically at room temperature for 12 h before incubation. A series of batches of MJYPV medium (Sako et al., 2003) were inoculated with samples of sandy sediment and the pH was adjusted to 8.0 with NaOH. Some of the batches of MJYPV medium containing these samples became turbid after 1 day of incubation at 70 °C. To obtain a pure culture, enriched cultures were streaked on to MJYPV plates containing 3 % (w/v) GP-700 agar (Shimizu Shokuhin), which remains solid at temperatures up to 80 °C, and were incubated at 70 °C. Well-defined colonies were selected and streaked onto fresh plates. This procedure was repeated at least three times. The isolate was designated strain KW1T and its purity was confirmed routinely by microscopy and by repeated partial sequencing of the 16S rRNA gene using PCR primers 27F and 1492R (Nakagawa et al., 2004).
Cells were observed using a differential interference microscope (Eclipse 800 system; Nikon). Negatively stained cells were examined by transmission electron microscopy, as described by Zillig et al. (1990); cells were stained with 1 % (w/v) uranyl acetate and examined using an H-700 electron microscope (Hitachi) at an accelerating voltage of 100 kV. The motile cells of strain KW1T were 1–4 µm in length and 0.3–0.8 µm in width. Electron microscopy of negatively stained samples revealed monopolar flagellation (Fig. 1). Cells stained Gram-positive during the early exponential phase and stained Gram-negative during the mid-exponential phase or stationary phase.
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; Brill & Wiegel, 1997; Nunoura et al., 2002).
Growth of the novel isolate under various conditions was determined by means of direct cell counting, after staining with 4',6-diamino-2-phenylindole (Porter & Feig, 1980), using an epifluorescence microscope (Eclipse 800 system; Nikon). We determined the effects of various temperatures on growth by cultivating the novel isolate in MJYPV medium (Sako et al., 2003). Strain KW1T grew at 52–78 °C, with an optimum at 70 °C. The strain did not grow at temperatures below 50 °C or above 80 °C. The effects of pH and salinity on growth of the isolate were therefore determined at 70 °C. The pH of MJYPV medium containing 20 mM MES (pH 5.0–6.0), PIPES (pH 6.3–7.0), HEPES (pH 7.3–8.0) or Tris (pH 8.5 and above) was adjusted with H2SO4 or NaOH at room temperature. After autoclaving, the pH was examined and readjusted aseptically with H2SO4 or NaOH at room temperature if necessary. The pH values of all media were stable during the cultivation period. The isolate grew at pH values between 5.5 and 9.0, with an optimum at pH 7.0. No growth was detected below pH 5.0 or above pH 9.5. To determine the effects of salinity on growth, the isolate was incubated in MJYPV medium containing various dilutions of 3x MJ synthetic seawater (Nakagawa et al., 2004) (1x MJ synthetic seawater contains 35 g sea salts). The isolate grew at 0–4.5 % (w/v) salinity, with an optimum at 1.0 %. Growth was not observed at salinities above 5.0 %. Growth of the isolate was inhibited completely in the presence of 0.1 % (w/v) Na2S2O3 in MJYPV medium (Sako et al., 1996a) at 70 °C. Other compounds such as 0.1 % (w/v) Na2SO3, Na2SO4 and cysteine hydrochloride did not affect growth of the isolate, whereas 3 % S0 inhibited growth completely.
We examined the growth of the novel isolate under anaerobic conditions. Air was replaced with N2 (100 kPa) and cells were applied to 5 ml MJYPV medium in the presence or absence of an electron acceptor such as 0.1 % (w/v) NaNO3, Na2SO3, NaNO2 or Na2S2O3. The strain did not grow under any of the tested anaerobic conditions.
We supplemented MJ synthetic seawater (Sako et al., 2003) with 0.2 and 0.02 % (w/v) of the following: yeast extract, trypticase peptone, yeast extract and trypticase peptone in combination, gelatin (Sigma), casein (Sigma), Casamino acids (Difco), starch (Wako), D(+)-glucose, sucrose, lactose, maltose, galactose, mannitol, fructose, cellulose, cellobiose, xylose, tartrate, acetate, butyrate, citrate, aminobutyric acid, pyruvate, succinate, malate, lactate, inositol, glycine, isoleucine, valine, serine, proline, lysine, arginine, histidine, threonine, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, glutamate, aspartate and cysteine. Strain KW1T grew heterotrophically on complex proteinaceous substrates such as yeast extract and trypticase peptone (alone or in combination), gelatin and Casamino acids. The strain also proliferated in the presence of acetate, pyruvate, succinate, malate, butyrate, proline, glycine, methionine, threonine, alanine, glutamine, glutamate and aspartate, but not in the presence of any of the sugars tested. The substrate concentrations (0.2 and 0.02 %) did not influence the results of these tests.
Genomic DNA of strain KW1T, Thermaerobacter marianensis JCM 10246T, Thermaerobacter nagasakiensis JCM 11223T and Thermaerobacter subterraneus ATCC BAA-137T was isolated as described by Lauerer et al. (1986) and the G+C content was determined by HPLC (Tamaoka & Komagata, 1984) using a DNA-GC kit (Yamasa Shouyu). The G+C content of strain KW1T was 70.8 mol%.
The 16S rRNA gene was amplified by means of a PCR using primers 27F and 1492R (Nakagawa et al., 2004) and the PCR product was sequenced directly by deoxynucleotide chain termination using an ABI 373A automated DNA sequencer (Applied Biosystems). The 16S rRNA gene sequence was aligned with a subset of 16S rRNA gene sequences obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and 1400 bases from each organism were analysed by neighbour joining (Saitou & Nei, 1987) using BIONJ (Gascuel, 1997). The 16S rRNA gene sequence of strain KW1T was very similar to those of other Thermaerobacter species (97.3–98.4 % similarity), but was not similar to those of any other organisms (Fig. 2).
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. Relatedness values were measured under optimal conditions (Tm–25 °C). The DNA of strain KW1T yielded relatively low hybridization signals with DNA from T. marianensis, T. nagasakiensis and T. subterraneus (38, 44 and 46 % relatedness, respectively). These findings indicated that the novel isolate could be differentiated genotypically from other Thermaerobacter species.
Members of the genus Thermaerobacter have been isolated from both hot marine and terrestrial environments. For example, T. marianensis and T. nagasakiensis were respectively isolated from the deep Mariana Trench (Takai et al., 1999) and from fluids in shallow hydrothermal vents in Tachibana Bay, Nagasaki Prefecture, Japan (Nunoura et al., 2002) and T. subterraneus was isolated from a terrestrial environment in the Great Artesian Basin of Australia (Spanevello et al., 2002). The levels of similarity between the 16S rRNA gene sequences of strain KW1T and other Thermaerobacter strains were found to be high, but the phenotypes were quite different (Table 1). For example, strain KW1T is motile with flagella, whereas T. marianensis and T. subterraneus are neither motile nor flagellated. Strain KW1T showed variable Gram-staining, whereas T. subterraneus is Gram-positive; electron microscopic examination of thin sections showed that the latter has a cell wall lacking the outer cell membrane that is typical of Gram-negative species (Spanevello et al., 2002). These organisms also differed in terms of the utilization of carbon sources and in the effects of pH and phosphate on growth. Several carboxylic acids support the growth of strain KW1T but not that of either T. nagasakiensis or T. subterraneus. Thiosulfate inhibited the growth of strain KW1T, but not that of other Thermaerobacter species. Notably, the growth of T. nagasakiensis is stimulated by S0 and thiosulfate. Finally, DNA–DNA hybridization indicated that strain KW1T is genotypically different from other Thermaerobacter species. On the basis of these physiological and genetic properties, we propose that this organism represents a novel species, for which we propose the name Thermaerobacter litoralis sp. nov.
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Cells are Gram-reaction-variable, motile, flagellated rods 1–4 µm long and 0.3–0.8 µm wide. Forms colonies on agar plates without forming spores and grows at 52–78 °C (optimum, 70 °C) and pH 5.5–9.0 (optimum, pH 7.0) in the presence of 0–4.5 % NaCl (optimum 1.0 %). Growth is heterotrophic in the presence of proteinaceous complex substrates, gelatin, Casamino acids, acetate, pyruvate, succinate, malate, butyrate, proline, glycine, methionine, threonine, alanine, glutamine, glutamate and aspartate. Growth is inhibited by thiosulfate and S0. The G+C content of the genomic DNA of the type strain is 70.8 mol% (HPLC).
The type strain, KW1T (=JCM 13210T=DSM 17372T), was isolated from a coastal hydrothermal beach on the Satsuma peninsula, Kagoshima Prefecture, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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