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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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ABSTRACT |
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The GenBank accession numbers for the 16S rRNA gene sequences of strains AB04T and K4T are AF001963 and AY248711.
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MAIN TEXT |
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). Pikuta et al. (2000) first described the type species of the genus as an obligate anaerobe. Later, Pikuta et al. (2003) corrected the description of the species A. pushchinoensis from ‘obligate anaerobe’ to ‘aerotolerant anaerobe’ and also changed the description of the genus Anoxybacillus from one comprising obligate anaerobes to facultative anaerobes to one comprising aerotolerant anaerobes or facultative anaerobes. At the time of writing, the genus Anoxybacillus contained three species: Anoxybacillus flavithermus, A. pushchinoensis Pikuta et al. 2000 and Anoxybacillus gonensis Belduz et al. 2003. The present paper describes the isolation, morphology, biochemical profile, 16S rRNA gene sequence and results of DNA–DNA hybridization with close relatives of two facultatively anaerobic, moderately thermophilic, facultatively alkaliphilic isolates that represent novel species of the genus Anoxybacillus. Strain AB04T (pH range 6·0–11·0) is proposed as the type strain of Anoxybacillus ayderensis sp. nov. Strain K4T (pH range 6·0–10·5) is proposed as the type strain of Anoxybacillus kestanbolensis sp. nov.
Isolation of strains
Two strains of Gram-positive rods, strains AB04T and K4T, were isolated from mud and water samples of the Ayder and Kestanbol hot springs in the provinces of Rize and Canakkale, respectively, in Turkey. The water temperature of these hot springs is around 60–70 °C. After collection, mud and water samples were immediately used for enrichment in nutrient broth (NB) at 60–70 °C. One-day-old enrichment cultures were repeatedly subcultured in 10 ml NB and streaked on agar plates to obtain separate colonies. The purity of the isolates was assessed by using colony morphology and microscopy. After 48 h growth on nutrient agar medium, colonies of strain AB04T were 1–2 mm in diameter, cream, regular in shape with round edges. Colonies of strain K4T were the same except that they were 1–2·5 mm in diameter. Light microscopy revealed that cells of strains AB04T and K4T were motile and were, respectively, 0·55x4·60 µm and 0·65x4·75 µm in size.
Biochemical and nutritional characteristics
The utilization of organic compounds as sole carbon sources was tested in basal medium (5 ml) supplemented with 0·5 % (w/v) concentrations of the following compounds (which had been separately sterilized as stock solutions): D-glucose, D-mannitol, D-mannose, D-sucrose, D-xylose, L-arabinose, D-fructose, lactose, D-raffinose, starch and L-rhamnose. Incubation was carried out at 60 °C. Strains AB04T and K4T were nutritionally versatile and used a wide variety of carbohydrates when grown on basal medium. Strain AB04T grew on D-glucose, D-raffinose, D-sucrose, D-xylose, D-fructose, L-arabinose, maltose and D-mannose, while strain K4T grew on D-mannitol, D-glucose, D-fructose, maltose, D-mannose, D-raffinose and D-sucrose (Table 1). Anaerobic growth was tested in anaerobic agar medium. Strains AB04T and K4T grew well aerobically but are facultatively anaerobic bacteria. Strain K4T grew well on anaerobic agar medium without yeast extract but strain AB04T grew on anaerobic agar medium only when supplemented with yeast extract.
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; strains AB04T and K4T were catalase- and oxidase-positive.
Salt and antibiotic sensitivity
Four sets of NB were prepared containing NaCl at 1, 1·5, 2, 2·5, 3, 4, 5 and 7 %. The growth of the isolates at different salt concentrations was tested using NB as organic substrate and a control broth without any NaCl supplementation. Growth of strain AB04T and growth of strain K4T were inhibited in the presence of NaCl concentrations above 2·5 and 4·0 %, respectively, and in the presence of ampicillin (25 µg ml–1), streptomycin sulphate (25 µg ml–1), tetracycline (12·5 µg ml–1), gentamicin (10 µg ml–1) and kanamycin (10 µg ml–1). The optimal NaCl concentrations for growth of AB04T and K4T were 1·5 and 2·5 %, respectively.
Spore formation
The formation of spores was tested for by using microscopic observation of both liquid cultures and single colonies of the isolates from agar plates at different incubation periods. Incubation periods of 1–2 days were required before spore formation became detectable on agar plates. Light microscopy revealed that strains AB04T and K4T were sporulating bacilli. Cells of strains AB04T and K4T formed terminal, spherical endospores.
SDS-PAGE analysis
Extraction of proteins from growing cells, measurement of protein concentrations in the extracts, electrophoresis and staining of proteins bands were performed as described previously (Belduz et al., 2003). The electrophoretic patterns of the soluble cellular proteins, as determined by the PAGE method (Fig. 1), showed that AB04T and K4T are not similar to A. flavithermus DSM 2641T, A. pushchinoensis DSM 12423T, A. gonensis NCIMB 13933T or each other (Fig. 1).
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). On the basis of 16S rRNA gene sequence analysis, AB04T has more than 98 % similarity to the sequences of A. gonensis NCIMB 13933T and A. flavithermus DSM 2641T, and has 97 % similarity to A. pushchinoensis DSM 12423T. The 16S rRNA gene sequence of K4T exhibits 97 % similarity to that of A. flavithermus DSM 2641T and more than 96 % sequence similarity to A. gonensis NCIMB 13933T and A. pushchinoensis DSM 12423T.
16S rRNA gene sequences obtained from the GenBank database were aligned and a neighbour-joining phylogenetic tree was constructed by using DNADIST and NEIGHBOR programs implemented as part of the PHYLIP package (Felsenstein, 1993). Phylogenetic analysis revealed a clustering of K4T and AB04T in the same branch with other Anoxybacillus species (Fig. 2).
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). The G+C contents of strains AB04T and K4T are 54 and 50 mol%, respectively, which are lower than those of A. flavithermus DSM 2641T and A. gonensis NCIMB 13933T.
On the basis of 16S rRNA gene sequence analysis, isolates AB04Tand K4T showed 
97 % similarity to other Anoxybacillus species; therefore, a DNA–DNA hybridization study was performed among AB04T, K4T, Anoxybacillus flavithermus DSM 2641T, Anoxybacillus gonensis NCIMB 13933T and Anoxybacillus pushchinoensis DSM 12423T. Isolation of genomic DNA for DNA–DNA hybridization and determination of DNA–DNA hybridization were performed as described previously (Belduz et al., 2003). DNA–DNA hybridization performed between AB04T and A. gonensis NCIMB 13933T revealed 68·6 % relatedness. However, strain AB04T differs from A. gonensis NCIMB 13933T in its growth temperature range and optimum, pH range and optimum, NaCl tolerance, reduction of nitrate to nitrite and utilization of some sugars as carbon source. Thermophilic isolate K4T showed similarity to A. flavithermus DSM 2641T, but DNA–DNA hybridization performed between K4T and A. flavithermus DSM 2641T showed only 60·4 % relatedness.
As the novel isolates were found to be closely related genetically to A. gonensis NCIMB 13933T and A. flavithermus DSM 2641T, we decided that they belong to the genus Anoxybacillus. The genus contains another species, A. pushchinoensis. In this study, DNA–DNA hybridization revealed 45·1 % DNA–DNA relatedness between AB04T and A. pushchinoensis and 42·9 % DNA–DNA relatedness between K4T and A. pushchinoensis. On the basis of DNA–DNA hybridization, strains AB04T and K4T show 40·5 % relatedness (Table 2). Wayne et al. (1987) suggested that relatedness levels below 70 % indicate that strains belong to different species.
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). The FAME profiles of AB04Tand K4T show that the main fatty acid is C15 : 0 iso (48·17 and 68·62 %, respectively); C15 : 0 iso is also the main fatty acid in A. gonensis, A. pushchinoensis and A. flavithermus. AB04T, K4T, A. flavithermus DSM 2641T and A. gonensis NCIMB 13933T contain C17 : 0 iso as a major fatty acid, but A. pushchinoensis DSM 12423T differs by having C16 : 0 as a major component. This indicates that there are similarities among the FAME profiles of Anoxybacillus species, including strains AB04T and K4T, which also share approximately 97 % 16S rRNA sequence similarity.
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Description of Anoxybacillus ayderensis sp. nov.
Anoxybacillus ayderensis (ay.de.ren'sis. N.L. masc. adj. ayderensis pertaining to Ayder, a hot spring in the province of Rize, Turkey, where the type strain was isolated).
Cells are Gram-positive, motile, spore-forming rods, 0·55x4·60 µm in size. Terminal, spherical endospores are formed. Colonies are 1–2 mm in diameter, cream, regular in shape with round edges. Catalase- and oxidase-positive. Starch and gelatin are hydrolysed. D-Glucose, D-raffinose, D-sucrose, D-xylose, D-fructose, L-arabinose, maltose and D-mannose are utilized. Nitrate is reduced to nitrite. Urease, indole and H2S are not produced. Growth occurs in the absence of NaCl; optimum growth at 1·5 % NaCl. No growth at concentrations above 2·5 % NaCl. The pH range for growth is 6·0–11·0; optimum pH is 7·5–8·5. Growth is inhibited in the presence of ampicillin (25 µg ml–1), streptomycin sulphate (25 µg ml–1), tetracycline (12·5 µg ml–1), gentamicin (10 µg ml–1) and kanamycin (10 µg ml–1). The temperature range for growth is 30–70 °C; optimum growth at 50 °C. Facultative anaerobe. DNA G+C content is 54 mol% (by melting temperature).
The type strain, AB04T (=NCIMB 13972T=NCCB 100050T), was isolated from Ayder Hot Spring, Turkey.
Description of Anoxybacillus kestanbolensis sp. nov.
Anoxybacillus kestanbolensis (kes.tan.bo.len'sis. N.L. masc. adj. kestanbolensis pertaining to Kestanbol, a hot spring in the province of Canakkale, Turkey, where the type strain was isolated).
Cells are Gram-positive, motile, spore-forming rods, 0·65x4·75 µm in size. Terminal, spherical endospores are formed. Colonies are 1–2·5 mm in diameter, cream, regular in shape with round edges. Catalase- and oxidase-positive. Starch is hydrolysed but gelatin is not. D-Mannitol, D-glucose, D-fructose, maltose, D-mannose, D-raffinose and D-sucrose are utilized. Nitrate is reduced to nitrite. Urease, indole and H2S are not produced. Growth occurs in the absence of NaCl; optimum growth at 2·5 % NaCl. No growth at concentrations above 4 % NaCl. The pH range for growth is 6·0–10·5; optimum pH is 7·5–8·5. Growth is inhibited in the presence of ampicillin (25 µg ml–1), streptomycin sulphate (25 µg ml–1), tetracycline (12·5 µg ml–1), gentamicin (10 µg ml–1) and kanamycin (10 µg ml–1). The temperature range for growth is 40–70 °C; optimum at 50–55 °C. Facultative anaerobe. DNA G+C content is 50 mol% (by melting temperature).
The type strain, K4T (=NCIMB 13971T=NCCB 100051T), was isolated from Kestanbol Hot Spring, Turkey.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1974). Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge: Cambridge University Press.
Felsenstein, J. (1993). PHYLIP (Phylogenetic Inference Package), version 3.6a3. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G. & Laurinavichius, K. (2000). Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. Int J Syst Evol Microbiol 50, 2109–2117.[Abstract]
Pikuta, E., Cleland, D. & Tang, J. (2003). Aerobic growth of Anoxybacillus pushchinoensis K1T: emended descriptions of A. pushchinoensis and the genus Anoxybacillus. Int J Syst Evol Microbiol 53, 1561–1562.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
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