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Karadeniz Technical University, Faculty of Arts and Sciences, Department of Biology, 61080 Trabzon, Turkey
Correspondence
Ali Osman Belduz
belduz{at}ktu.edu.tr
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Published online ahead of print on 31 January 2003 as DOI 10.1099/ijs.0.02473-0.
The GenBank accession number for the 16S rRNA gene sequence of Anoxybacillus gonensis G2T is AY122325.
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). Over the years, a number of spore-forming thermophiles have been reported, mainly in the genera Bacillus and Clostridium (Guagliardi et al., 1996). In this study, we isolated some thermophilic bacilli from the Gonen and Diyadin hot springs, respectively located in the Turkish provinces of Balikesir and Agri. On the basis of preliminary experiments, a representative strain appeared to differ from other thermophilic bacilli with respect to the utilization of xylose; it was therefore characterized further. Xylose isomerase is an intracellular enzyme that catalyses the conversion of D-xylose to D-xylulose. Its practical significance stems from its ability to isomerize D-glucose to D-fructose. Therefore, this enzyme is often referred to as glucose isomerase and is widely used in industry for the production of high-fructose corn syrup.
The present paper describes the isolation, morphological, physiological and biochemical profiles and 16S rRNA sequence of this strain and the results of DNA–DNA hybridization with close relatives and proposes that it represents a novel species of the genus Anoxybacillus (Pikuta et al., 2000), Anoxybacillus gonensis sp. nov.
Isolation of strains
Seven Gram-positive rods were isolated from mud and water samples from the Gonen and Diyadin hot springs. The water temperature of these hot springs is around 70 °C. After collection, mud and water samples were used immediately for enrichment in nutrient broth at 60–70 °C. One-day-old enrichment cultures were repeatedly subcultured in 10 ml nutrient broth and streaked on agar plates to obtain separate colonies. The purity of the isolates was assessed by colony morphology and microscopy. After 48 h growth on nutrient agar medium, colonies of strain G2T were small, cream, irregularly shaped with rough edges and 2–3 mm in diameter. Light microscopy revealed that cells of the strain were rod-shaped, Gram-positive and motile, measuring 0·75x5·0 µm.
Biochemical and nutritional characteristics
Utilization of organic compounds as sole carbon sources was tested in basal medium (5 ml) supplemented with 0·5 % (w/v) of the following compounds, which had been separately sterilized as stock solutions: glucose, mannitol, mannose, sucrose, xylose, arabinose, lactose, raffinose, starch, glycogen and rhamnose. Incubation was carried out at 60 °C. The strain was nutritionally versatile and used a wide variety of carbohydrates when grown on basal medium. It grew on glucose, glycogen, raffinose, sucrose, xylose and mannitol (Table 1). Anaerobic growth was tested in anaerobic agar medium. Strain G2T grew well aerobically, but was facultatively anaerobic.
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. Strain G2T is catalase- and oxidase-positive.
Salt and antibiotic sensitivity
Four replicate sets of nutrient broth were prepared containing 1, 2, 3, 4, 5 or 7 % NaCl. Growth of the isolate at different salt concentrations was tested using nutrient broth as the organic substrate and using a control broth without any NaCl supplementation. Growth was inhibited in the presence of NaCl concentrations above 4 % and in the presence of chloramphenicol (25 µg ml-1), ampicillin (25 µg ml-1), streptomycin sulphate (25 µg ml-1) and tetracycline (12·5 µg ml-1). The optimal NaCl concentration for growth was 2 %.
Spore formation
The formation of spores was tested by microscopic observation of both liquid cultures and single colonies of the isolates from agar plates after different incubation periods. Incubation for 1–2 days was required before spore formation became detectable on agar plates. Light microscopy revealed that strain G2T was a sporulating bacillus. It formed terminal spherical endospores.
16S rRNA gene sequence analysis
The 16S rRNA gene was selectively amplified from purified genomic DNA by using oligonucleotide primers designed to anneal to conserved positions in the 3' and 5' regions of bacterial 16S rRNA genes. The forward primer, UNI16S-L (5'-ATTCTAGAGTTTGATCATGGCTTCA-3'), corresponded to positions 11–26 of the Escherichia coli 16S rRNA, while the reverse primer, UNI16S-R (5'-ATGGTACCGTGTGACGGGCGGTGTTGTA-3'), corresponded to the complement of positions 1411–1393 of E. coli 16S rRNA (Brosius et al., 1978). PCR conditions were according to Beffa et al. (1996). The PCR product was cloned into pGEM-T and then the 16S rRNA gene sequence was determined with an Applied Biosystems model 373A DNA sequencer, using the ABI PRISM cycle-sequencing kit. A sequence consisting of about 1400 nt of the 16S rRNA gene of strain G2T was determined. The sequence was compared with the 16S rDNA sequences of some representatives of the Bacillus group by using PHYLIP version 3.5 (Felsenstein, 1989). Phylogenetic analysis revealed a clustering with Anoxybacillus flavithermus DSM 2641T (97 % sequence similarity). These sequences differed by 7–16 % from sequences of species of the genus Bacillus and can therefore be distinguished as a separate genus. The sequence of strain G2T showed 96, 93 and 86 % similarity, respectively, to sequences from Anoxybacillus pushchinoensis DSM 12423T, Saccharococcus caldoxylosilyticus DSM 12041T and Alicyclobacillus acidocaldarius.
PCR amplification of intergenic 16S–23S rDNA sequences
Primers FGPS1490-72 (5'-TGCGGCTGGATCCCCTCCTT-3'; positions 1521–1541 of the E. coli 16S rRNA gene sequence) and FGPL132'-38 (5'-CCGGGTTTCCCCATTCGG-3'; positions 114–132 of the E. coli 23S rRNA gene sequence) were used for amplification of intergenic 16S–23S rDNA sequences. PCR conditions were according to Riffard et al. (1998). As shown in Fig. 1, all seven novel strains showed a faint band of about 300 bp, but only A2, A6 and G2T had the same pattern, with A4, A5, A7 and A9 showing a different banding pattern. However, the internally transcribed spacer (ITS) patterns of all the novel strains were different from those of Anoxybacillus flavithermus DSM 2641T and S. caldoxylosilyticus DSM 12041T; therefore, G2T is different from these strains.
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), the G+C content was determined from the denaturation temperature in 0·5x SSC. Denaturation profiles were followed at 260 nm using a thermoprogrammable spectrophotometer (Jenway 6105 UV/Vis spectrophotometer) in accordance with the principles of Mandel & Marmur (1968). The G+C content of this strain is 57 mol%, which is lower than that of Anoxybacillus flavithermus DSM 2641T.
DNA was isolated by chromatography on hydroxyapatite. DNA–DNA hybridization was determined at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983) and Escara & Hutton (1980). A Gilford System model 2600 spectrophotometer equipped with a Gilford model 2527-R thermoprogrammer and plotter was used. Renaturation rates were computed with the TRANSFER.BAS program (Ahmad et al., 2000). DNA–DNA hybridization studies were performed among G2T, A4, A7 and Anoxybacillus flavithermus DSM 2641T and between G2T and Anoxybacillus pushchinoensis DSM 12423T (Table 2).
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. The protein concentration in the extracts was measured according to the method of Bradford (1976) and 40 µg crude extract was loaded per lane. Electrophoresis on 12 % SDS-PAGE was carried out as described by Laemmli (1970). Proteins were stained in a solution that contained Coomassie brilliant blue R-250 (0·125 %), methanol (50 %) and acetic acid (10 %) for 2–4 h and then visualized by destaining in a solution of 5 % methanol/7 % acetic acid. The electrophoretic patterns of soluble cellular proteins, as determined by SDS-PAGE (Fig. 2), showed that G2T is not similar to Anoxybacillus flavithermus DSM 2641T, Anoxybacillus pushchinoensis DSM 12423T or S. caldoxylosilyticus DSM 12041T, and G2T therefore does not belong to any of these species. The other six novel strains were examined; SDS-PAGE analysis showed the similarity of these strains to G2T (Fig. 2).
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; the fatty acid profiles of the novel strains were very similar, with C15 : 0 iso as the main fatty acid (62–68 %). Strain G2T also resembled Alicyclobacillus acidocaldarius on the basis of fatty acid profiles, but the morphological and biochemical properties and 16S rRNA gene sequence of G2T do not resemble those of this bacterium.
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. Xylose isomerase activity was then measured as described by Lee et al. (1990). The enzyme activity was determined as 0·78 and 0·5 U (mg protein)-1 at 60 °C on xylose and glucose, respectively, from crude cell lysate.
Initial studies indicated that G2T was a member of Bacillus cluster 5, as defined by Ash et al. (1991). On the basis of genotypic and phenotypic properties, the isolate can be distinguished from other Bacillus species described. Despite the morphological observations that clearly class this bacterium as a Bacillus, its closest relative, on the basis of 16S rRNA sequence analysis, is Anoxybacillus flavithermus DSM 2641T.
Stackebrandt & Goebel (1994) reached the conclusion that strains belonging to the same genus that exhibit less than 97 % 16S rRNA gene sequence similarity should be considered members of different species. However, it is also known that analysis of 16S rRNA sequences may be insufficient to distinguish between some species (Vandamme et al., 1996). In this study, we determined the 16S rRNA gene sequence of G2T and found more than 97 % similarity to that of Anoxybacillus flavithermus. However, we also determined that some physiological, morphological and biochemical characteristics of our isolate differ from those of Anoxybacillus flavithermus DSM 2641T. In addition, Daffonchio et al. (1998) showed that the 16S–23S ITS of Bacillus cereus are well conserved in terms of length; in contrast, bacilli such as Bacillus licheniformis and Bacillus subtilis have at least two different ITS fingerprints. In this study, we showed that G2T has a different ITS fingerprint from Anoxybacillus flavithermus DSM 2641T and S. caldoxylosilyticus DSM 12041T. As a result of the ITS study, we suggest that our isolate is different from Anoxybacillus flavithermus.
On the basis of 16S rRNA sequence analysis, these thermophilic isolates resemble Anoxybacillus flavithermus, but a DNA–DNA hybridization study performed between G2T and Anoxybacillus flavithermus showed that this isolate is only 53·4 % similar to Anoxybacillus flavithermus. Since the novel isolate was found to be closely related genetically to Anoxybacillus flavithermus, we conclude that our novel isolates belong to the genus Anoxybacillus. The genus has one other species, Anoxybacillus pushchinoensis. In this study, we found 45 % similarity between G2T and Anoxybacillus pushchinoensis on the basis of DNA–DNA hybridization. Wayne et al. (1987) suggested that strains of a species show more than 70 % DNA–DNA relatedness, indicating that strain G2T and Anoxybacillus pushchinoensis represent different species.
On the basis of these data, we suggest that our thermophilic isolate (G2T) is not related to either Anoxybacillus flavithermus DSM 2641T or Anoxybacillus pushchinoensis DSM 12423T at the species level (in view of threshold value of 70 % recommended by Wayne et al., 1987), and we propose that strain G2T should be placed in the genus Anoxybacillus as the type strain a novel species, Anoxybacillus gonensis sp. nov.
On the basis of their morphological, physiological, biochemical and fatty acid profiles and 16S rRNA sequences, the other six novel isolates are strains of Anoxybacillus gonensis. Although there are some differences between A4, A5, A7 and G2T in terms of ITS patterns and total protein profiles, DNA–DNA hybridization also indicated that all seven novel isolates are strains of Anoxybacillus gonensis (Table 2
).
Description of Anoxybacillus gonensis sp. nov.
Anoxybacillus gonensis (gon.en'sis. N.L. masc. adj. gonensis pertaining to Gonen, a hot spring in the province of Balikesir, Turkey, where the type strain was isolated).
Cells are rod-shaped, Gram-positive, motile and spore-forming, 0·75x5·0 µm. Forms terminal spherical endospores. Colonies are rough and cream in colour. Weakly catalase-positive. Oxidase-positive. Starch and gelatin are hydrolysed. Glucose, glycogen, raffinose, sucrose, xylose, fructose and mannitol are utilized. Nitrate is not reduced to nitrite. Urease, indole and H2S are not produced. Grows in 4 % NaCl broth. The pH range for growth is 6·0–10·0 (optimum pH 7·5–8·0). The temperature range for growth is 40–70 °C (optimum 55–60 °C). Facultative anaerobe. The G+C content of the DNA is 57 mol% (by melting temperature).
The type strain, G2T (=NCIMB 13933T=NCCB 100040T), was isolated from Gonen hot spring, Turkey.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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