J. Gen. Appl. Microbiol., 54, 327 334 (2008)
Full Paper Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov., a thermophilic bacterium isolated from a hot spring in Batman Reyhan Gul-Guven,1 Kemal Guven,1, * Annarita Poli,2 and Barbara Nicolaus2, * 1
Department of Biology, Faculty of Science, Dicle University, 21280, Diyarbakir, Turkey
2 Istituto di Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078, Pozzuoli, (Napoli) Italy
(Received July 15, 2008; Accepted July 29, 2008)
A new thermophilic spore-forming strain KG8T was isolated from the mud of Taslidere hot spring in Batman. Strain KG8T was aerobe, Gram-positive, rod-shaped, motile, occurring in pairs or filamentous. Growth was observed from 35 65 C (optimum 55 C) and at pH 5.5 9.5 (optimum pH 7.5). It was capable of utilizing starch, growth was observed until 3% NaCl (w/v) and it was positive for nitrate reduction. On the basis of 16S rRNA gene sequence similarity, strain KG8T was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone―menaquinone-7; major fatty acid―iso-C15:0 and iso-C17:0) supported the affiliation of strain KG8T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain KG8T. Based on these results we propose assigning a novel subspecies of Anoxybacillus kamchatkensis, to be named Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. with the type strain KG8T (DSM 18475T=CIP 109280T). Key Words——Anoxybacillus; DNA-DNA hybridization; lipid; taxonomy; thermophile; 16S rRNA analysis
Introduction
The genus Anoxybacillus was first described by Pikuta et al. (2000); the genus comprises ten species, Anoxybacillus flavithermus (Pikuta et al., 2000), Anoxy * Address reprint requests to: Prof. Kemal Guven, Department of Biology, Faculty of Science, Dicle University, 21280, Diyarbakir, Turkey. Tel. +90 412 2488550 (x.3064) Fax. +90 412 2488039 E-mail:
[email protected]；and Barbara Nicolaus, Istituto di Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078, Pozzuoli, (Napoli) Italy. Tel. +390818675245 Fax. +390818041770 E-mail:
[email protected] The EMBL accession number for the 16S rRNA sequence of Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. is AM999779.
bacillus pushchinoensis (Pikuta et al., 2003), Anoxybacillus gonensis (Belduz et al., 2003), Anoxybacillus contaminans (De Clerck et al., 2004), Anoxybacillus voinovskiensis (Yumoto et al., 2004), Anoxybacillus ayderensis and Anoxybacillus kestanbolensis (Dulger et al., 2004), Anoxybacillus kamchatkensis (Kevbrin et al., 2005), Anoxybacillus amylolyticus (Poli et al., 2006) and Anoxybacillus rupiensis (Derekova et al., 2007). Pikuta et al. (2003) corrected the description of the genus Anoxybacillus from one comprising obligate anaerobes to facultative anaerobes to one comprising aerotolerant anaerobes or facultative anaerobes. Some thermophilic bacilli from the mud of Taslidere hot spring in Batman, located in Turkey s southeast province were isolated. On the basis of preliminary experiments, a representative strain appeared to differ from other thermophilic bacilli with respect to the utili-
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zation of carbohydrates. The present paper describes the isolation, morphological, physiological, biochemical profiles and 16S rRNA sequence of this strain belonging to Anoxybacillus species. The characteristics of this isolate are compared with its near neighbor and on the basis of DNADNA hybridization values and biochemical properties we propose that it represents a novel subspecies of Anoxybacillus kamchatkensis. Materials and Methods
Location of sampling. Isolates were collected from the mud of Taslidere hot spring in Batman, a city in the southeast of Turkey. The temperature and pH of the muddy water were 78 C and 6.7, respectively. The order of chemicals in the water from most to the least are SO4 ＞ Cl ＞ HCO3 and Ca ＞ Na ＞ K. The class of hot spring water was with Ca, Na, SO4 and Cl. Culture medium and growth condition. All samples were immediately incubated in the temperature range 35 65 C in different enrichment media. They were: (% value are in w/v) medium (A) containing yeast extract Oxoid (varying from 0.3 to 0.6%) and NaCl (0.3 0.6%) at pH 5.6 and at pH 4.0; medium (B) yeast extract Oxoid 0.4%, (NH4)2SO4 0.2%, KH2PO4 0.3%, 4 ml Solution A and 4 ml Solution B at pH 3.4, 4.0 and 6.0 (Solution A=125 g/L (NH4)2SO4 plus 50 g/L MgSO4・7 H2O; Solution B=62.5 g/L CaCl2・2H2O); medium (C) Nutrient Broth (Oxoid). Cultures were purified from the samples grown in enrichment medium C at 55 C, pH 7.5, using the serial dilution technique, repeated at least five times. Several isolates were selected and taxonomic properties of strain KG8T will be presented in this paper. Anoxybacillus contaminans DSM 15866T and Geobacillus toebii DSM 14590T, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Brunschweig, Germany (DSMZ) and were grown according to the DSMZ catalogue. Anoxybacillus gonensis NCIMB13933T, Anoxybacillus ayderensis NCIMB13972T and Anoxybacillus voinovskiensis NCIMB 13956T were obtained from the National Collections of Industrial, Marine and Food Bacteria, Scotland, UK (NCIMB). Anoxybacillus kamchtkensis DSM 14988T was kindly provided by Vadim V. Kevbrin and grown as reported by Kevbrin et al. 2005. Morphological and physiological tests. The temperature range for growth was determined by incubat-
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ing the isolate from 37 to 75 C. The pH dependence of growth was tested in the pH range of 4.0 to 9.5. Growth on single carbon source was tested on liquid medium C diluted 1:10 (v/v) in tap water at pH 7.5. The organic compounds (1%, w/v) were: D-glucose, D-lactose, D-maltose, D-fructose, D-arabinose, D-cellobiose, D-galactose, D-mannose, D-ribose, D-trehalose, D-xylose, L-sorbose, raffinose, glycerol, sucrose, and sodium acetate. All growth tests were done at 55 C and growth was scored positive if the A540 was greater than 0.300 after 3 days. Cellular morphology was determined by phase-contrast microscopy (Zeiss). Colony morphology was determined by a Leica M8 stereomicroscope using cultures grown on media A agar (2%) plates for 24 h at 55 C. Gram staining was performed according to Dussault (1955). KOH test was performed according to Halebian et al. (1981). Unless otherwise stated the strain was characterized using the modified methods of Gordon and Pang (1973). Starch hydrolysis was tested by flooding cultures on solid enrichment medium A containing 0.2% (w/v) starch with Lugol s iodine. For casein hydrolysis, solid medium A plus an equal quantity of skimmed milk was used. For gelatine hydrolysis and sensibility to lysozyme, enrichment medium A plus 1.0% (w/v) gelatine or 0.001% (w/v) lysozyme was used, respectively. For the spore formation test, enrichment medium A plus 0.001% (w/v) MnCl2・4H2O was used. Tolerance of NaCl was determined in medium A with varying concentrations of NaCl. Urease activity was determined as described by Lanyi (1987). Aminopeptidase was assayed with the Bactident Aminopeptidase Kit of Merck (Germany). Hydrolysis of N -benzoyl-arginine-p-nitroanilide (BAPA) stereoisomer was tested according to Oren and Galinski (1994). All tests were carried on cells grown in the enrichment medium A at 55 C. An anaerobic growth test was performed as described by Schäffer et al. (2004). Antimicrobial susceptibility testing. Sensitivity of the strain to the antibiotics was tested by using the enrichment-solid medium A and sensi-discs (6 mm, Oxoid). The following antibiotics were used (μg): neomycin (5, 30), erythromycin (30), penicillin G (10 U), nystatin (100), chloramphenicol (10, 50), kanamycin (5, 30), tylosin (10, 30), ampicillin (25), gentamicin (10, 30), novobiocin (30), bacitracin (10 U), lincomycin (15), fusidic acid (10), vancomycin (30), streptomycin (25) and tetracycline (30, 50).
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Lipid and fatty acid compositions. Lipid and fatty acids analysis were performed as reported by Nicolaus et al. (2001). Polar lipids were achieved from 3.0 g of freeze-dried cells grown in enrichment medium C at 55 C, by the extraction with 65:25:4 CHCl3/MeOH/H2O. The lipid extract was analyzed by thin layer chromatography (TLC) on silica gel (0.25 mm, F254, Merck) eluted with CHCl3/MeOH/H2O (65:25:4 by vol.). Lipids were detected by spraying the plates with 0.1% Ce(SO4)2 in 2 N H2SO4 followed by heating at 100 C for 5 min. Phospholipids and aminolipids were detected on the plates upon spraying with the Dittmer-Lester and the ninhydrin reagents respectively and glycolipids were visualized with α-naphtol. Authentic standards were used for comparing the Rf of substances. Fatty acid methyl esters (FAMEs) were obtained from complex lipids by acid methanolysis (Nicolaus et al., 2001) and analyzed using a Thermo GC Polaris Q gas chromatograph fitted with a FID detector and equipped with an HP-V column with a flow rate of 1 ml/min using the temperature program of 170 C (3 min), from 170 to 270 C at 5 C/min. Identification of compounds was obtained with standards and by interpretation of mass spectra. Compounds were identified by 1H-NMR and EI/MS (Electronic Impact) as previously described (Nicolaus et al., 2001). NMR spectra, recorded at the NMR Service of Institute of Biomolecular Chemistry of CNR (Pozzuoli, Italy), were acquired on a Bruker DPX-300 operating at 300 MHz, using a dual probe. Phylogenetic analysis. Genomic DNA extraction, PCR mediated amplification of the 16S rDNA and purification of the PCR products were carried out as described previously (Rainey et al., 1994, 1996). The primers for 16S rDNA amplification were: 10-30F 5 GAGTTTGATCCTGGCTCAG and 1500R 5 AGAAAGGAGGTGATCCAGCC. The resulting 16S rRNA sequence was manually aligned and compared with representative sequences of organisms belonging to the Firmicutes group (Maidak et al., 1999). The results are presented as a phylogenetic tree. The 16S rRNA gene similarity values were calculated by pairwise comparison of the sequences within the alignment. For construction of the phylogenetic dendrogram, operations of the PHYLIP package were used (Felsenstein, 1993); the pair wise evolutionary distances were computed from percent similarities by the correction of Jukes and Cantor (1969) and the phylogenetic tree was constructed by the neighbor-joining method (Saitou and
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Nei, 1987). The root of the tree was determined by including the 16S rRNA gene sequence of Brevibacillus centrosporus into the analysis. The 16S rRNA gene sequence of KG8T strain was submitted to EMBL Nucleotide Sequence Database and has been the assigned accession number of AM999779. DNA-DNA hybridization. DNA-DNA hybridization was performed according to Sunna et al. (1997). DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostated 6×6 multicell changer and a temperature controller with in situ temperature probe (Varian). Results
Morphological and biochemical analysis Cells of isolate KG8T were Gram-positive, motile rods, 2.0 2.5 μm long and 0.5 μm wide (Fig. 1), occurring in pairs or filamentous with terminal, ellipsoidal to cylindrical endospores. Colonies were usually cream, smooth and circular. KG8T strain was an aerobe that grew in Nutrient Broth Medium (C) and in enrichment medium A. The temperature growth range was from 35 to 65 C with an optimum growth at 55 C. The pH growth range was
Fig. 1. Phase-contrast microscopy of strain KG8T at stationary phase, grown on medium C at 55 C.
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Table 1. Comparison of the phenotypic characteristics of strain KG8T and related species. Characteristic Relation to O2
Temperature range ( C) Optimum temperature ( C) pH range Optimum pH Motility NaCl (3%, w/v) Nitrate reduction Sensitive to Lysozyme Gelatin hydrolysis Starch hydrolysis Casein hydrolysis Oxidase Catalase Utilization of Glucose Galactose Lactose Xylose Trehalose
1 aerobe
35 65
2
3
4
5
6
7
aerobe/ faculta- aerobe/ faculta- aerobe/ anaerfacultative an- faculta- tive an- faculta- obe/ aerobe tive an- aerobe tive an- faculta- tive anaerobe tive an- aerobe aerobe aerobe 38 67 30 72 40 70 50 60 30 64 45 65
55
57 62
60 65
5.5 9.5 7.5 + + +
5.7 9.9 6.8 8.5 + +* +* +*
5.5 9.0 6.0 10.0 4.5 10.0 7.0 8.0 7.0 7.5 8.0 7.0 7.0 8.0 + + +/ + + + + + + nd nd nd nd
+ +
*
+ + + +
+ + nd + w
+ + +
+ +
+ +
w
+ + +
nd
+ nd + nd
+ + + + +
+ + + +
+ +
nd
55 60
50
54
61
8
9
10
11
faculta- facultative antive aerobe anaerobe
anaerobe
aerobe
37 66
35 67
30 70
40 70
62
55
50
50 55
5.0 6.5 8.0 10.5 5.5 8.5 6.0 11.0 6.0 10.5 5.6 9.5 9.7 6.0 6.5 7.5 8.5 7.5 8.5 + + + + + + + + + + nd nd nd nd nd
+ + nd +
+ + nd + +
+ nd + +
+ nd nd + +
+ + nd
+ nd + nd
+ nd nd
1, strain KG8T; 2, A. kamchatkensis DSM 14988T (Kevbrin et al., 2005); 3, A. flavithermus DSM 2641T (Pikuta et al., 2000); 4, A. gonensis NCIMB 13933T (Belduz et al., 2003); 5, A. contaminans DSM 15866T (De Clerck et al., 2004); 6, A. voinovskiensis NCIMB 13956T (Yumoto et al., 2004); 7, A. amylolyticus DSM 15939T (Poli et al., 2006); 8, A. pushchinoensis DSM 12423T (Pikuta et al., 2003); 9, A. rupiensis DSM 17127T (Derekova et al., 2007); 10, A. ayderensis NCIMB 13972T and 11, A. kestanbolensis NCIMB 13971T(Dulger et al., 2004). + positive ; negative ; w weak response ; n.d. not determined. * data obtained by present paper.
from 5.5 to 9.5 with an optimum at pH 7.5. The isolate KG8T utilized, as carbon sources, D-galactose and sodium acetate weakly. The isolate was sensitive to penicillin G (10 U), chloramphenicol (10 μg), kanamycin (5 μg), tylosin (10 μg), lincomycin (15 μg), bacitracin (10 U), gentamicin (10 μg), novobiocin (30 μg), fusidic acid (10 μg), streptomycin (25 μg), tetracycline (30 μg) and ampicillin (10 μg) and resistance to nystatin (100 μg). The isolate KG8T was catalase and starch hydrolysis positive while it was negative for oxidase, gelatine hydrolysis, and casein-hydrolysis. Isolate KG8T was able to hydrolyze hippurate, to decompose tyrosine, to reduce nitrate and was not sensitive to lysozyme (Table 1).
Lipid and fatty acid composition The isolate KG8T strain possessed complex lipids based on fatty acids. The total lipid content ranged between 8 and 10% of the total dry weight of cells grown at 55 C in medium C. Under these conditions three major phospholipids, 1,2 diacylglycero-3-phosphorylethanolamine (PEA), 1,2 diacylglycero-3-phosphorylglycerol (PG), and cardiolipin (DPG) and one minor unknown glycolipid were identified (Fig. 2). For comparison to KG8T strain, Geobacillus toebii DSM 14590T and Anoxybacillus kamchatkensis DSM 14988T, grown as described above, were analyzed. FAMEs composition, determined on cells grown under standard condition, was characterized by the abundance of branched acyl chains. The most abun-
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Fig. 2. Thin-layer chromatogram on Merck silica gel F254 of total polar lipids from strain KG8T (lane 3) and reference strains Anoxybacillus amylolyticus DSM 15939T (lane 1), Geobacillus toebii DSM 14590T (lane 2), Anoxybacillus kamchatkensis DSM 14988T (lane 4). The solvent system was chloroform/methanol/water (65:25:4 by vol.). A) Phospholipids were detected by spraying the plate with Dittmer and Lester reagent; UK unknown phospholipids, UK2 unknown phospho-glycolipid, PEA 1,2 diacylglycero-3-phosphorylethanolamine, PG 1,2 diacylglycero-3-phosphoryl-glycerol, DPG cardiolipin. B) Glycolipids were detected by spraying the plate with α-naphthol reagent; UK1 unknown glycolipid, UK2 unknown phospho-glycolipid. Table 2. Fatty acid methyl ester profiles of KG8T strain and Anoxybacillus speciesa. Fatty acids
1
2
3
4
5
6
7
8
9
10
11
iC13:0 iC14:0 C14:0 C14:0 i3-OH iC15:0 aiC15:0 C15:0 iC16:0 aiC16:0 C16:0 iC17:1ω5c iC17:0 C17:0 anteiso A aiC17:0 C17:0 aiC17:1 C17:1 iC18:0 aiC18:0 C18:0
― ― 1.4 ― 40.5 7.7 0.6 11.7 0.5 9.3 ― 16.5 ― 11.7 ― ― ― ― ― ―
― 0.9 1.6 ― 9.1 7.7 2.6 20.7 0.4 21.0 ― 9.3 ― 18.9 2.6 ― ― 2.0 ― 3.0
― ― ― ― 41.2 2.1 0.1 7.0 0.1 6.3 ― 31.6 ― 8.4 0.7 ― ― 0.1 0.7 1.9
― ― 3.0 ― 52.0 7.0 ― 5.0 ― 11.0 ― 12.0 ― 7.0 ― ― ― ― ― ―
― 1.3 1.3 ― 54.7 8.0 ― 7.1 ― 1.9 ― 3.9 ― ― ― 7.1 2.6 ― ― ―
― ― 1.9 ― 54.8 4.0 1.2 2.9 ― 11.1 ― 17.7 ― 6.2 ― ― ― ― ― ―
― 1.2 1.2 ― 65.2 2.6 1.1 6.0 ― 2.4 2.6 12.0 0.8 3.3 ― ― ― ― ― ―
― ― 2.9 ― 51.9 7.5 ― 5.1 ― 11.3 ― 11.6 ― 7.0 ― ― ― ― ― ―
― ― 0.3 ― 53.0 1.6 0.3 2.0 ― 5.4 ― 33.6 ― 3.9 ― ― ― ― ― ―
― ― 1.0 ― 48.2 3.6 0.8 7.5 ― 9.1 ― 20.6 ― 9.2 ― ― ― ― ― ―
― 0.9 1.3 ― 68.6 3.5 1.1 6.4 ― 3.5 0.6 9.5 ― 3.7
― ― ― ―
1, strain KG8T; 2, A. kamchatkensis DSM 14988T (data obtained by present paper); 3, A. amylolyticus DSM 15939T (Poli et al., 2006); 4, A. contaminans DSM 15866T (De Clerck et al., 2004); 5, A. voinovskiensis NCIMB 13956T (Yumoto et al., 2004); 6, A. flavithermus DSM 2641T (Pikuta et al., 2000); 7, A. gonensis NCIMB 13933T (Belduz et al., 2003); 8, A. pushchinoensis DSM 12423T (Pikuta et al., 2003); 9, A. rupiensis DSM 17127T (Derekova et al., 2007); 10, A. ayderensis NCIMB 13972T and 11, A. kestanbolensis NCIMB 13971T (Dulger et al., 2004). aValues are shown as a percentage of the total fatty acid content for each strain.
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Fig. 3. Phylogenetic tree, based on 16S rRNA gene sequences, showing the relationship between the members of the genus Anoxybacillus. Bootstrap values (%) are based on 1,000 replicates and are shown at each node. Bar, 0.01 expected changes per site.
dant was iC15:0 (40.5%), other components were iC17:0 (16.5%), aiC17:0 (11.7%), iC16:0 (11.7%), nC16:0 (9.3%), aiC15:0 (7.7%), nC14:0 (1.45%) and nC15:0 (0.63%) (Table 2). Chromatographic analysis of quinones revealed the presence of one more abundant UV-absorbing band. 1H-NMR spectrum showed that the quinone present is of the menaquinone (MK) type. The LC/MS analysis gave a molecular peak corresponding to MK7 as the major compound (more than 90% of quinones).
The average is 67.5%. This value was borderline with the recommended threshold value of 70% which is accepted as the definition of a genospecies (Wayne et al., 1987). Therefore more evidence, such as lipid and fatty acid analysis, carbon sources, antibiotic sensitivity and other biochemical features, had been required to establish KG8T strain as a new subspecies of Anoxybacillus kamchatkensis.
Phylogenetic analysis and DNA-DNA hybridizations The total 16S rRNA gene sequence of the strain KG8T (EMBL nucleotide sequence accession number is AM999779) showed high similarity (99%) to Anoxybacillus kamchatkensis (98%) to Anoxybacillus gonensis, Anoxybacillus flavithermus and Anoxybacillus ayderensis. The bacterium shared a similarity of 97%, and 94% with Anoxybacillus pushchinoensis and Anoxybacillus kestanbolensis, respectively. The results are presented as a phylogenetic dendrogram (Fig. 3). KG8T was separate from other species within the Bacillus radiation, including the phylogenetically related Anoxybacillus species (De Clerck et al., 2004). The DNA-DNA reassociation values found between strain KG8T and the close strains Anoxybacillus gonensis and Anoxybacillus ayderensis were 48% and 42%, respectively. In addition the DNA-DNA hybridization between strain KG8T and Anoxybacillus kamchatkensis were performed in triplicate (65.1%, 66.7% and 70.7%).
This is the first study that has been carried out on the isolation of thermophilic bacteria in the Taslidere hot spring (Batman, Turkey). The strain KG8T is a moderate thermophilic Grampositive rod, aerobe that shows phenotypic features in common with the other thermophilic species isolated from geothermal soil. The total 16S rRNA sequence of strain KG8T falls within the radiation of the genus Anoxybacillus. The genotypic and phenotypic properties of strain KG8T differ from those of the known species of this genus. The analysis of the almost complete nucleotide sequence of 16S rRNA of strain KG8T allowed a phylogenetic tree to be constructed, with the closest neighbors of related species (Fig. 3). Strain KG8T is closely related to the species A. kamchatkensis. It has been largely accepted that if the level of 16S rRNA similarity is greater than 97% additional phenotypic and genotypic characteristics should be used for taxonomic purpos-
Discussion
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es. We have also compared our microorganism with the other Anoxybacillus species. Table 1 indicates the difference in the phenotypic and biochemical characteristics between strain KG8T and its closest relatives. Strain KG8T differed from the other relatively closely related species in terms of the following phenotypic characteristics: morphology, optimal growth temperature, pH range for growth, catalase and oxidase reactions, reduction of NO3 to NO2, utilization of substrates, starch hydrolysis, relation to O2 (Table 1). A comparison of the polar lipid patterns by one-dimensional TLC revealed high levels of similarity between KG8Tand Anoxybacillus amylolyticus but the lipid pattern of KG8T strain differed from Anoxybacillus kamchatkensis for types and relative quantities of phospholipids and for glycolipid presence (Fig. 2). The high level of branched chain (iC15:0 and iC17:0) in the fatty acid composition of strain KG8T confirmed the affiliation to the genus Anoxybacillus (Table 2). Fatty acid profiles of KG8T strain and Anoxybacillus kamchatkensis were also different in respect to iC15:0 percentage (Table 2). The borderline DNA-DNA hybridization between strain KG8T and Anoxybacillus kamchatkensis (67.5%) didn t allow us to differentiate KG8T from all the other species of this genus into a separate species (Wayne et al., 1987). On the basis of lipid analyses, fatty acid profile, biochemical and physiological properties, we propose that Anoxybacillus strain KG8T is a novel subspecies of Anoxybacillus kamchatkensis, for which the name Anoxybacillus kamchatkensis subsp. asaccharedens is proposed. Description of Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. A. kamchatkensis subsp asaccharedens (a.sac.cha. re dens. Gr. pref. a-, not; L. n. saccharon, sugar; L. part. adj. edens, consuming, devouring; N.L. part. adj. asaccharedens, not consuming sugars, not using carbohydrates as sole carbon sources). Cells are Gram-positive, sporulating rods (2.0 2.5 μm long and 0.5 μm wide), occurring in pairs or filamentous with terminal, ellipsoidal to cylindrical endospores. Motile. Colonies on Nutrient Broth medium (Oxoid) are circular, cream, and smooth. It is thermophilic and aerobe, exhibiting optimum growth temperature of 55 C, but is able to grow between 35 and 65 C and at pH 7.5. It is able to utilize D-galactose and sodium acetate weakly, when the medium is supplemented with 0.06%
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yeast extract. Negative reactions for D-glucose, D-lactose, D-fructose, D-arabinose, D-cellobiose, D-mannose, D-ribose, D-xylose, L-sorbose, glycerol, and as carbon sources were obtained. It is positive for catalase, tyrosine decomposition, hippurate hydrolysis, starch hydrolysis and nitrate reduction, negative with respect to casein hydrolysis, not sensitive to lysozyme, gelatine hydrolysis, or oxidase. MK7 is the most abundant menaquinone and the predominant polar lipids are 1,2 diacylglycero-3-phosphorylethanolamine (PEA), 1,2 diacylglycero-3-phosphoryl-glycerol (PG), cardiolipin (DPG) and a minor glycolipid. iC15:0, iC17:0, iC16:0, aiC17:0, are the main cellular fatty acids. The following antibiotics inhibited the growth of KG8T: penicillin G, chloramphenicol, kanamycin, tylosin, lincomycin, bacitracin, gentamicin, novobiocin, fusidic acid, streptomycin, tetracycline and ampicillin and it was resistant to nystatin. KG8T was isolated from the mud of Taslidere hot spring in Batman (Turkey). Type strain KG8T has been deposited in Centre de Ressources Biologiques de I Institut Pasteur (CIP 109280T) and in Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSM 18475T). EMBL Nucleotide Sequence Database accession number is AM999779. Acknowledgments The paper was supported by Scientific Research Commission of Dicle University-Diyarbakir (Projects; DUAPK-04-FF-42 and DUAPK-04-FF-45). The authors thank Eduardo Pagnotta for technical assistance, Dominique Melck for NMR-ICB service and Dr. Ilaria Finore, PhD student, for the growth of Anoxybacillus species. We are grateful to Dr. J.P. Euzeby, who kindly helped with the correct epithet of the new subspecies. References Belduz, A. O., Dulger, S., and Demirbag, Z. (2003) Anoxybacillus gonensis sp. nov., a moderately thermophilic, xyloseutilizing, endospore-forming bacterium. Int. J. Syst. Evol. Microbiol., 53, 1320 1351. Cashion, P., Hodler-Franklin, M. A., McCully, J., and Franklin, M. (1977) A rapid method for base ratio determination of bacterial DNA. Anal. Biochem., 81, 461 466. De Clerck, E., Rodriguez-Diaz, M., Vanhoutte, T., Heyrman, J., Logan, N. A., and De Vos, P. (2004) Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov. isolated from contaminated gelatin batches. Int. J. Syst. Evol. Microbiol., 54, 941 946. De Ley, J., Cattoir, H., and Reynaerts, A. (1970) The quantitative
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measurement of DNA hybridization from renaturation rates. Eur. J. Biochem., 12, 133 142. Derekova, A., Sjoholm, C., Mandeva, R., and Kambourova, M. (2007) Anoxybacillus rupiensis sp. nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria). Extremophiles, 11, 577 583. Dulger, S., Demirbag, Z., and Belduz, A. O. (2004) Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov. Int. J. Syst. Evol. Microbiol., 54, 1499 1503. Dussault, H. P. (1955) An improved technique for staining redhalophilic bacteria. J. Bacteriol., 70, 484 485. Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package), version 3.5.1. Department of Genome Sciences, University of Washington, Seattle, USA. Gordon, R. H. W. C. and Pang, C. H. N. (1973) The genus Bacillus (Agricult. Monogr. 427), Department of Agriculture, Washington, DC, USA. Halebian, S., Harris, B., Finegold, S. M., and Rolfe, R. D. (1981) Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria. J. Clin. Microbiol., 13, 444 448. Huss, V. A. R., Festl, H., and Schleifer, K. H (1983) Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol., 4, 184 192. Jukes, T. H. and Cantor, C. R. (1969) Evolution of protein molecules. In Mammalian Protein Metabolism, ed. by Munro, H. N., Academic Press, New York, pp. 21 132. Kevbrin, V. V., Zengler, K., Lysenko, A. M., and Wiegel, J. (2005) Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka. Extremophiles, 9, 391 398. Lanyi, B. (1987) Classical and rapid identification methods for medically important bacteria. Methods Microbiol., 19, 1 67. Maidak, B. L., Cole, J. R., Parker, C. T., Jr., Garrity, G. M., Larsen, N., Li, B., Lilburn, T. G., McCaughey, M. J., Olsen, G. J., Overbeek, R., Pramanik, S., Schmidt, T. M., Tiedje, J. M., and Woese, C. R. (1999) A new version of the RDP (Ribosomal Database Project). Nucleic. Acids Res., 27, 171 173. Nicolaus, B., Manca, M. C., Lama, L., Esposito, E., and Gambacorta, A. (2001) Lipid modulation by environmental stresses in two models of extremophiles isolated from Antarctica. Polar Biol., 24, 1 8. Oren, A. and Galinski, E. A. (1994) Hydrolysis of N -benzoylarginine-p-nitroanilide stereoisomers as a phenotypic test: A study of Gram-positive halotolerant bacteria. Syst. Appl. Microbiol., 17, 7 10 Pikuta, E., Cleland, D., and Tang, J. (2003) Aerobic growth of Anoxybacillus pushchinoensis K1T: Emended descriptions of A. pushchinoensis and the genus Anoxybacillus. Int. J.
Vol. 54
Syst. Evol. Microbiol., 53,1561 1562. Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G., and 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. Poli, A., Esposito, E., Lama, L., Orlando, P., Nicolaus, G., de Appolonia, F., Gambacorta, A., and Nicolaus, B. (2006) Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). Syst. Appl. Microbiol., 29, 300 307. Rainey, F. A., Fritze, D., and Stackebrandt, E. (1994) The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis. FEMS Microbiol. Lett., 115, 205 212. Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M., and Stackebrandt, E. (1996) The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage; proposal of Nocardiopsaceae fam. nov. Int. J. Syst. Bacteriol., 46, 1088 1092. Saitou, N. and Nei, M. (1987) The neighbour-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406 425. Schäffer, C., Franck, W. L., Scheberl, A., Kosma, P., McDermott, T. R., and Messner, P. (2004) Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov. Int. J. Syst. Evol. Microbiol., 54, 2361 2368. Stackebrandt, E. and Swiderski, J. (2002) From phylogeny to systematics: The dissection of the genus Bacillus. In Applications and Systematics of Bacillus and Relatives, ed. by Berkeley, R., Heyndrickx, M., Logan, N. A., and De Vos, P., Blackwell Publishing, Oxford, pp. 8 22. Sunna, A., Tokajian, S., Burghardt, J., Rainey, F., Antranikian, G., and Hashwa, F. (1997) Identification of Bacillus kaustophilus, Bacillus thermocatenulatus and Bacillus strain HSP as members of Bacillus thermoleovorans. Syst. Appl. Microbiol., 20, 232 237. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., Starr, M. P., and Trüper, H. G. (1987) Report of the Ad Hoc Committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol., 37, 463 464. Yumoto, I., Hirota, K., Kawahara, T., Nodasaka, Y., Okuyama, H., Matsuyama, H., Yokota, Y., Nakajima, K., and Hoshino, T. (2004) Anoxybacillus voinovskiensis sp. nov., a moderately thermophilic bacterium from a hot spring in Kamchatka. Int. J. Syst. Evol. Microbiol., 54, 1239 1242.
