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Gram-Positive Bacteria

Bacillus indicus sp. nov., an arsenic-resistant bacterium isolated from an aquifer in West Bengal, India

K. Suresh, S. R. Prabagaran, S. Sengupta, S. Shivaji

  • 1Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India
  • 2Geological survey of India, GSI Complex, DK-6, Sector-II, Bidhannagar, Kolkata - 700 091, India
  • Correspondence
    S. Shivaji
    shivas{at}ccmb.res.in

    • International Journal of Systematic and Evolutionary Microbiology 2004; 54(4):1369–1375 · https://doi.org/10.1099/ijs.0.03047-0

    View at publisher PubMed

    Abstract

    Strain Sd/3T (=MTCC 4374T=DSM 15820T), an arsenic-resistant bacterium, 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). The bacterium was Gram-positive, rod-shaped, non-motile, endospore-forming and yellowish-orange pigmented. It possessed all the characteristics that conform to the genus Bacillus, such as it had A4β 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.

    • Published online ahead of print on 9 February 2004 as DOI 10.1099/ijs.0.03047-0.

    • The GenBank accession number for the 16S rRNA gene sequence of strain Sd/3T is AJ583158.

    The ground-water and soil of the Bengal basin in India contains high levels of arsenic (Karim, 2000; 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 3×105 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 (×1000) 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 A4β 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 A4β 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⇓⇓.

    Table 1.

    Phenotypic characteristics that differentiate B. indicus sp. nov. (Sd/3T) from related Bacillus species

    Strains: 1, B. indicus Sd/3T (present study); 2, B. cohnii DSM 6307T (present study); 3, B. megaterium MTCC 1684 (present study); 4, B. flexus DSM 1320T (present study); 5, B. arseniciselenatis DSM 15340T (Blum et al., 1998); 6, B. selenitireducens DSM 15326T (Blum et al., 1998); 7, B. niacini IFO 15566T (Nagel & Andreesen, 1991); 8, B. pumilus ATCC 7061T (Venkateswaran et al., 2003; Nagel & Andreesen, 1991); 9, Bacillus firmus ATCC 14575T (Venkateswaran et al., 2003; Nagel & Andreesen, 1991); 10, B. psychrosaccharolyticus ATCC 23296T (Larkin & Stokes, 1967); 11, Bacillus alcalophilus DSM 485T (Fritze et al., 1990; Nielsen et al., 1995; Yumoto et al., 2003); 12, Bacillus clarkii DSM 8720T (Nielsen et al., 1995); 13, Bacillus halmapalus DSM 8723T (Logan et al., 2002; Nielsen et al., 1995); 14, B. horikoshii DSM 8719T (Li et al., 2002; Logan et al., 2002; Nielsen et al., 1995); 15, Bacillus krulwichiae IAM 15000T (Yumoto et al., 2003); 16, Bacillus okuhidensis JCM 10945T (Li et al., 2002); 17, Bacillus bataviensis LMG 21833T (Heyrman et al., 2004); 18, Bacillus veddri DSM 9768T (Agnew et al., 1995); 19, Bacillus luciferensis LMG 18422T (Logan et al., 2002). +, Positive; −, negative; r, resistant; s, sensitive; nd, no data available.

    Table 2.

    Fatty acid composition (%) of B. indicus sp. nov. (Sd/3T) and related Bacillus species

    Strains: 1, B. indicus Sd/3T (present study); 2, B. cohnii DSM 6307T (present study); 3, B. megaterium MTCC 1684 (present study); 4, B. flexus DSM 1320T (present study); 5, B. bataviensis LMG 21833T (Heyrman et al., 2004); 6, B. pumilus ATCC 7061T (Kämpfer, 1994); 7, B. firmus ATCC 14575T (Kämpfer, 1994); 8, B. krulwichiae IAM 15000T (Yumoto et al., 2003); 9, B. okuhidensis JCM 10945T (Li et al., 2002); 10, B. luciferensis LMG 18422T (Logon et al., 2002); 11, B. alcalophilus JCM 5262T (Yumoto et al., 2000). Fatty acid data for B. arseniciselenatis, B. niacini, B. psychrosaccharolyticus, B. clarkii, B. halmapalus, B. horikoshii, B. veddri and B. selenitireducens were not reported in the relevant references. B. indicus Sd/3T (1), B. cohnii (2), B. megaterium (3) and B. flexus (4) were grown in nutrient broth at 30 °C for the determination of fatty acid composition in the present study.

    Bacillus cohnii DSM 6307T and Bacillus flexus DSM 1320T, obtained from DSMZ, Germany, and Bacillus megaterium MTCC 1684, obtained from Institute for Microbial Technology, Chandigarh, India, were used as reference strains in studies related to morphology, motility, biochemical tests, identification of fatty acids and DNA–DNA hybridization. DNA–DNA hybridization was performed by the membrane filter method (Tourova & Antonov, 1987) 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 A4β 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.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on 16S rDNA (1473 bases) sequences showing the phylogenetic relationship between B. indicus sp. nov. (Sd/3T) and other related species of the genus Bacillus and related reference micro-organisms. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are given at the nodes.

    Description of Bacillus indicus sp. nov.

    Bacillus indicus (in′di.cus L. masc. adj. indicus pertaining to India, Indian).

    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 A4β-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.

    Acknowledgments

    Thanks to the Department of Biotechnology, Government of India, New Delhi, India and the Indo-French Centre for Promotion of Advanced Research, New Delhi, India, for the facilities. The authors would also like to thank Dr G. S. N. Reddy, Centre for Cellular and Molecular Biology, Hyderabad, India for suggestions and his interest in the work.

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