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1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India
2 Geological survey of India, GSI Complex, DK-6, Sector-II, Bidhannagar, Kolkata - 700 091, India
Correspondence
S. Shivaji
shivas{at}ccmb.res.in
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murein type (L-orn-D-Asp) peptidoglycan variant, MK-7 as the major menaquinone and iso-C15 : 0 and anteiso-C15 : 0 as the major fatty acids. Based on its chemotaxonomic and phylogenetic characteristics, strain Sd/3T was identified as a species of the genus Bacillus. It exhibited maximum similarity (95 %) at the 16S rRNA gene level with Bacillus cohnii; however, DNA–DNA similarity with B. cohnii was 60·7 %. Strain Sd/3T also exhibited a number of phenotypic differences from B. cohnii (DSM 6307T). These data suggest that Sd/3T represents a novel species of the genus Bacillus. The name Bacillus indicus sp. nov. is proposed.
The GenBank accession number for the 16S rRNA gene sequence of strain Sd/3T is AJ583158.
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; Das et al., 1996). The presence of arsenic in the environment may thus lead to the enrichment of arsenic-resistant bacteria. Bacteria belonging to the genera Acidithiobacillus, Bacillus, Deinococcus, Desulfitobacterium and Pseudomonas have already been reported to be resistant to arsenic (De Vicente et al., 1990; Sato & Kobayashi, 1998; Dopson et al., 2001; Niggemyer et al., 2001; Prithivirajsingh et al., 2001; Suresh et al., 2004). In the current study, we report on the isolation and characterization of an arsenic-resistant bacterium from an arsenic contaminated aquifer located in the Bengal basin in India.
Source of the organisms, media and growth conditions
Strain Sd/3T was isolated from a sand sample obtained from an arsenic-contaminated aquifer in Chakdah district in West Bengal, India (23° 3' N 88° 35' E) that yielded about 3x105 c.f.u. (g soil)–1. The medium used for isolation was nutrient agar (0·5 % peptone, 0·5 % NaCl, 0·2 % yeast extract, 0·2 % beef extract and 1·5 % agar, pH 7·0, all w/v) containing 5 % (w/v) sodium arsenate. Nutrient agar without sodium arsenate was used for growth and maintenance.
Morphology, motility and biochemical and chemotaxonomic characteristics
Sd/3T was grown in nutrient broth and examined by phase-contrast microscopy (x1000) to ascertain cell shape and the presence of motility. Biochemical tests were performed on cultures grown at 30 °C in Luria–Bertani broth (1·0 % tryptone, 0·5 % yeast extract, 0·9 % NaCl, pH 7·2, all w/v). Hydrolysis of aesculin, nitrate reduction, indole test, Voges–Proskauer test, methyl red test and H2S production were performed as described by Lanyi (1987). Utilization of various carbon compounds, hydrolysis of casein, gelatin, starch and Tween 20 were performed as described by Smibert & Krieg (1994). Oxidase activity was qualitatively detected using the method described by Stanier et al. (1966). Further, the ability of the cultures to utilize carbon compounds as the sole carbon source was checked by supplementing minimal medium [1·05 % K2HPO4, 0·45 % KH2PO4, 0·1 % (NH4)2SO4 and 1·5 % agar, all w/v] with 0·5 % (w/v) of the filter-sterilized carbon compound. Nutrient agar was used to check the sensitivity of Sd/3T to different antibiotics, using commercial antibiotic discs (HiMedia, Mumbai, India).
Sd/3T was grown in nutrient broth at 30 °C for 48 h and the fatty acid methyl esters were extracted (Sato & Murata, 1988) and analysed as described previously (Reddy et al., 2002). Isoprenoid quinones were extracted according to the method described by Collins et al. (1977) and separated by HPLC using a SB-C18 Zorbax reverse-phase column fixed to a Hewlett Packard Series 1100 HPLC as described previously (Tamaoka et al., 1983; Suresh et al., 2004). Polar lipids were analysed as described by Suresh et al. (2004). Peptidoglycan type was analysed according to Komagata & Suzuki (1987). Mol% G+C content of the DNA was determined as described earlier (Shivaji et al., 1989; Reddy et al., 2000).
Phenotypic and chemotaxonomic characteristics of Sd/3T are described in the species description. It is apparent that Sd/3T, which is Gram-positive, rod-shaped, non-motile, endospore-forming and which possesses A4 murein type (L-orn-D-Asp) peptidoglycan variant (Schleifer & Kandler, 1972), MK-7 as the main respiratory quinone, iso-C15 : 0, anteiso-C15 : 0, iso-C14 : 0, iso-C16 : 0 and iso-C17 : 0 as the predominant fatty acids, phosphatidylglycerol, phosphatidylethanolamine and phosphatidyldiglycerol as the polar lipids and a DNA G+C content of 41·2 mol%, conforms to the characteristics of the genus Bacillus (Tables 1 and 2) (Kämpfer, 1994). To date, only a few Bacillus species with oval spores and A4 murein-type peptidoglycan have been reported (Spanka & Fritze, 1993). Phenotypic and chemotaxonomic characteristics that differentiate Sd/3T from related Bacillus species are listed in Tables 1 and 2.
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) as described by Shivaji et al. (1992) and Reddy et al. (2000).
Phylogenetic analysis
The 16S rDNA was amplified as described previously (Shivaji et al., 2000) and the PCR product was purified using the QIA quick PCR purification kit (Qiagen). Sequencing of purified 16S rDNA was performed using the ABI PRISM Big Dye Terminator cycle sequencing kit (Applied Biosystems). Purified amplicons were electrophoresed on an automated DNA sequencer (ABI PRISM model, 3700). The partial (1473 bp) 16S rDNA was aligned with relevant sequences contained in EMBL database using CLUSTAL W (Thompson et al., 1994). Pairwise evolutionary distances were calculated using the DNADIST program with the Kimura two-parameter model (Kimura, 1980). Phylogenetic trees were constructed by four different tree-making algorithms (UPGMA, KITSCH, FITCH and DNAPARS) in the PHYLIP software package (Felsenstein, 1993). The stability among the clades of a phylogenetic tree was assessed by taking 1000 replicates of the dataset and was analysed using the programs SEQBOOT, DNADIST, UPGMA and CONSENSE contained in the PHYLIP software package.
The phylogenetic analyses, based on 16S rRNA gene sequence data, indicated that Sd/3T is most closely related to species of the genus Bacillus (Fig. 1). Sd/3T exhibited a similarity of 94 % to B. megaterium MTCC 1684, B. flexus DSM 1320T and Bacillus psychrosaccharolyticus ATCC 23296T and 95 % to B. cohnii DSM 6307T, Bacillus pumilus TUT1009, Bacillus horikoshii DSM 8719T, Bacillus niacini IFO 15566T and Bacillus bataviensis LMG 21833T following BLAST analysis. In the present study comparisons were made between Sd/3T and only B. cohnii and B. megaterium because the former had a similar A4 murein-type (L-orn-D-Asp) peptidoglycan, and because the latter is a representative species of the genus. All the other species, which exhibited 94–95 % similarity at the 16S rRNA gene level, differ from Sd/3T in peptidoglycan type and other phenotypic characteristics (Table 2
). The 16S rRNA gene sequence of Sd/3T shows a deletion of 14 bases between bases 55–68 compared to B. cohnii DSM 6307T. Furthermore, at the DNA–DNA level, Sd/3T shares 60·7 % (mean of three replicate values of 58, 60 and 64) and only 34·3 % (mean of three replicate values of 32, 34 and 37) relatedness with B. cohnii DSM 6307T and B. megaterium MTCC 1684, respectively. Thus, based on the less than 95 % similarity between Sd/3T and the reported species of Bacillus at the 16S rRNA gene level, it is proposed to assign novel species status to Sd/3T. In addition, Sd/3T differs from both B. cohnii DSM 6307T and B. megaterium MTCC 1684 in that the colonies are yellowish-orange in colour and are unable to grow in the presence of 5 % (w/v) NaCl but can grow in the presence of 3 mM arsenite (As III) and 20 mM arsenate (As V). Additionally, Sd/3T is positive for arginine dihydrolase, does not hydrolyse Tween 20 and utilizes meso-erythritol, D-mannose, D-ribose and L-arginine (Table 2). Phenotypic characteristics that differentiate Sd/3T from related Bacillus species are listed in Table 2. Based on the phenotypic differences and the phylogenetic data it is proposed to classify Sd/3T as a novel species of the genus Bacillus, for which the name Bacillus indicus sp. nov. is proposed.
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Cells are aerobic, Gram-positive, non-motile rods measuring approximately 0·9–1·2 µm wide and 3·3–5·3 µm long. Strain Sd/3T has sub-terminal endospores in a slightly swollen sporangium. Colonies on nutrient agar are yellowish-orange pigmented, circular, raised, smooth, convex and 3·0–4·0 mm in diameter. The pigment in acetone exhibits three absorption maxima at 404, 428 and 451 nm, characteristic of carotenoids. Grows in the range of 15–37 °C (optimum 30 °C) but not at 40 °C. Grows between pH 6 and 7 and tolerates up to 2·0 % (w/v) NaCl. Positive for catalase, gelatinase, amylase, arginine dihydrolase and aesculin. Does not hydrolyse Tween 20 or urea. Does not reduce nitrate to nitrite and is negative for indole production, Voges–Proskauer test and citrate utilization. Utilizes D-cellobiose, meso-erythritol, inositol, lactose, D-melibiose, D-maltose, D-mannose, sucrose, L-rhamnose, D-ribose, raffinose, L-arginine, L-tryptophan and L-tyrosine but not arabinose, fumarate, glutarate, pyruvate, L-proline and L-serine as sole carbon sources. Sensitive to the antibiotics ampicillin (10 µg), chloramphenicol (30 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (30 µg), rifampicin (30 µg), streptomycin (10 µg) and tetracycline (30 µg). Resistant to amoxycillin (10 µg). The major fatty acids are iso-C14 : 0 (10·9 %), iso-C15 : 0 (33·5 %), anteiso-C15 : 0 (19·3 %), iso-C16 : 0 (11·0 %), C16 : 0 (5·9 %) and iso-C17 : 0 (10·8 %). The main proportion of the polar lipids consists of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major respiratory quinone is MK-7. The cell wall is an A4-murein with ornithine as the diamino acid and aspartic acid as the interpeptide bridge. The G+C content of DNA is 41·2 mol%. The type strain is Sd/3T (=MTCC 4374T=DSM 15820T), and was isolated from sand of an arsenic-contaminated aquifer in West Bengal, India.
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ACKNOWLEDGEMENTS |
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