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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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7cis) were the major fatty acids. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain Con a/3T was identified as a member of the genus Bacillus. It exhibited maximum similarity (97 %) at the 16S rRNA gene level with Bacillus barbaricus (DSM 14730T); however, the DNA–DNA relatedness value with B. barbaricus was 60 %. Strain Con a/3T also exhibited a number of phenotypic differences from B. barbaricus (DSM 14730T). Strain Con a/3T was therefore identified as representing a novel species of the genus Bacillus, for which the name Bacillus arsenicus sp. nov. is proposed. The type strain is Con a/3T (=MTCC 4380T=DSM 15822T=JCM 12167T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Con a/3T is AJ606700.
Scanning electron micrographs of the surface of a spheroidal concretion, a comparison of the lipid composition of B. arsenicus sp. nov. and B. barbaricus, and a neighbour-joining tree showing the phylogenetic relationships between B. arsenicus sp. nov. and other species of the genus Bacillus are available as supplementary material in IJSEM Online.
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; Laverman et al., 1995; Oremland et al., 2001) belonging to the genera Acidithiobacillus, Bacillus, Deinococcus, Desulfitobacterium and Pseudomonas (de Vicente et al., 1990; Sato & Kobayashi, 1998; Dopson et al., 2001; Niggemyer et al., 2001; Prithiviraj Singh et al., 2001; Suresh et al., 2004a, b) have been reported to be resistant to arsenic. In this study, we report the isolation of an arsenic-resistant novel species of Bacillus that was isolated from a bore well located in the Chakdah region of West Bengal, India. The bacterium, Con a/3T, is resistant to arsenate and arsenite. Based on its phenotypic characteristics, chemotaxonomic properties and 16S rRNA gene sequence, the strain was identified as representing a novel species of the genus Bacillus, for which the name Bacillus arsenicus sp. nov. is proposed.
Source of the organism, media and growth conditions
Strain Con a/3T was isolated from a concretion, which is an extra-appendage-like structure observed on arsenic ore, recovered from the sediment of a bore-hole (depth 45 m) located in the Chakdah region of West Bengal, India (23° 3' N, 88° 35' E). The medium used for isolation of the bacterium was arsenate medium [0·5 % (w/v) peptone, 0·5 % (w/v) NaCl, 0·2 % (w/v) yeast extract, 0·2 % (w/v) beef extract, 1·5 % (w/v) agar; pH 7·0], containing 5 % sodium arsenate. Nutrient agar without sodium arsenate was used for growth and maintenance of the strain. Bacillus barbaricus (DSM 14730T) was obtained from the DSMZ, cultured on nutrient agar or the appropriate medium, and used for comparison of phenotypic and chemotaxonomic characteristics.
Electron microscopy of the concretion and electron probe micro analyser (EPMA) study
Scanning electron microscopy was used to study the external and internal morphologies of the concretions and also to visualize the bacteria. For these purposes, the concretion was surface-sterilized with alcohol, crushed with pincers to reveal the interior, mounted on stubs, coated with gold, and observed immediately using a model 5-440, LEO electron microscope; a working distance of 19 mm and an accelerating voltage of 20 kVA, 300 pA were used. Under the scanning electron microscope, the concretion appeared as a spherical, pimpled structure with rhombohedral crystals on the surface (see Supplementary Figure A(a) in IJSEM Online), whereas the internal cavity had a ruffled surface associated with rod-like structures, which were probably bacteria (see Supplementary Figure A(b) in IJSEM Online). Studies on the chemical composition of the concretion using an EPMA (CAMECA SX 51 instrument at the Geological Survey of India Laboratory, Faridabad, India) indicated that the mineral associated with the concretion was siderite, made up of FeCO3 (25·75–49·58 %), MnCO3 (0·24–16·75 %), CaCO3 (2·01–12·23 %) and MgCO3 (0·15–0·74 %).
Morphology, motility, and biochemical and chemotaxonomic characteristics
Strain Con a/3T was also observed under a phase-contrast microscope (DIAPLAN; Leitz) using an oil-immersion objective (x100) to ascertain the shape and motility. The physiological tests were done as described by Claus & Berkeley (1986). For the various biochemical tests listed in Table 1 and used in the description of the species, the culture was grown at 30 °C in Luria–Bertani broth medium [1·0 % (w/v) tryptone, 0·5 % (w/v) yeast extract, 2·5 % (w/v) NaCl; pH 7·2], and the tests were performed as described by Lanyi (1987) and Smibert & Krieg (1994). To check for the level of resistance to arsenic, strain Con a/3T was grown at 25 °C in nutrient broth [1·0 % (w/v) peptone, 0·5 % (w/v) yeast extract, 0·5 % (w/v) NaCl)] with 1·0 % (w/v) glucose, and containing either 5, 10 or 20 mM arsenate (Na2HAsO4) or 0·25 or 0·5 mM arsenite (As2O3). Growth was monitored spectrophotometrically (at OD600) for a period of 72 h. In addition, the ability of strain Con a/3T to utilize a carbon compound as the sole carbon source was investigated by supplementing the minimal medium [1·05 % (w/v) K2HPO4, 0·45 % (w/v) KH2PO4, 0·1 % (w/v) (NH4)2SO4, 1·5 % (w/v) agar] with 0·5 % (w/v) filter-sterilized carbon compound. Nutrient agar was used to check the sensitivity of strain Con a/3T to various antibiotics using antibiotic discs supplied by Hi-Media Pvt. (Mumbai, India).
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) were extracted from cells grown in nutrient broth at 30 °C and analysed as described by Reddy et al. (2002). Isoprenoid quinones were extracted according to the method described by Collins et al. (1977), separated by HPLC and identified as described by Reddy et al. (2003). Peptidoglycan was prepared and analysed according to the method described by Komagata & Suzuki (1987). Isolation of DNA, determination of the mol% G+C content of the DNA and analysis of the phospholipids were performed as described previously (Shivaji et al., 1989). DNA–DNA hybridization was performed by using the membrane filter method (Tourova & Antonov, 1987), as described previously (Shivaji et al., 1992).
To ascertain whether bacteria were present within the cavity of the concretion, about 10 mg of the concretion sample was separated from the ore surface by using forceps, surface-sterilized and crushed. The sample was then suspended in 1 ml of sterile distilled water, and serially diluted and plated on nutrient agar containing 5 % sodium arsenate. Colonies were visible after 3 days; the colony count was 2·0x103 (g concretion)–1. The colonies were all cream in colour, circular, raised, smooth, convex and 1–2 mm in diameter (Table 1
). One of the colonies was purified and the strain was labelled as Con a/3. The bacterium was rod-shaped, as confirmed by both electron microscopy and phase-contrast microscopy.
The phenotypic and chemotaxonomic characteristics of strain Con a/3T are given in the species description (see below). Strain Con a/3T is a Gram-positive, motile, endospore-forming, rod-shaped organism. Diaminopimelic acid is the characteristic peptidoglycan diamino acid, MK-7 is the major respiratory quinone, and iso-C15 : 0 (10·6 %), anteiso-C15 : 0 (10·5 %), iso-C16 : 0 (19·0 %), C16 : 0 (9·6 %) and C16 : 1(7cis) (25·3 %) are the prominent fatty acids (Table 2). The lipids present are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and, in addition, two unidentified aminolipids, one unidentified aminophospholipid and one unknown phospholipid. B. barbaricus (DSM 14730T) has a similar lipid composition except that, instead of two unknown aminolipids, it has one aminolipid and one lipid (see Supplementary Figure B in IJSEM Online). The DNA G+C content of strain Con a/3T was 35·0 mol%, thus conforming to the characteristics of the genus Bacillus (Kämpfer, 1994).
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), and the PCR product was purified using the QIA quick PCR purification kit (Qiagen). Sequencing was performed by using an ABI PRISM model 3700 automatic DNA sequencer and the Big Dye Terminator cycle sequencing kit (both from Applied Biosystems). The almost complete 16S rRNA gene sequence (1505 bp) was aligned with closely related sequences retrieved from EMBL by using CLUSTAL W (Thompson et al., 1994). Pairwise evolutionary distances were computed by using the DNADIST program with the Kimura two-parameter model (Kimura, 1980). Phylogenetic trees were constructed by using NEIGHBOR, UPGMA, KITSCH, FITCH and DNAPARS of the PHYLIP package (Felsenstein, 1993). The stability among the clades of a phylogenetic tree was assessed by taking 1000 replicates of the dataset, which were analysed by using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package.
Phylogenetic analysis based on the 16S rRNA gene sequence, indicated that strain Con a/3T is closely related to B. barbaricus DSM 14730T (97 %); lower 16S rRNA gene sequence similarity was observed with other Bacillus species, including Bacillus flexus DSM 1320T (94 %), Bacillus cohnii DSM 6307T (94 %), Bacillus megaterium IAM 13418T (94 %), Bacillus bataviensis DSM 15601T (94 %), Bacillus niacini DSM 2923T (94 %), Bacillus novalis LMG 21837T (94 %), Bacillus benzoevorans DSM 5391T (94 %), Bacillus lentus NCIMB 8773T (94 %), Bacillus gelatini DSM 15865T (93 %), Bacillus naganoensis ATCC 53909T (93 %) and Bacillus indicus Sd/3T (91 %). The neighbour-joining phylogenetic tree further confirmed that strain Con a/3T is phylogenetically related to species of Bacillus and forms a clade with B. barbaricus (DSM 14730T), with a bootstrap value of 100 % (Fig. 1; also see Supplementary Figure C in IJSEM Online). Phylogenetic trees constructed by using KITSCH, FITCH, UPGMA and DNAPARS also yielded similar stable groupings.
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) at the DNA–DNA level. DNA–DNA similarity was not done with those species showing more than 3 % difference at the 16S rRNA gene sequence level. Therefore, it would appear that, based on the more than 3 % difference at the 16S rRNA gene sequence level and less than 70 % similarity at the DNA–DNA level with the closest related Bacillus species, strain Con a/3T can probably be classified as representing a novel species according to accepted criteria (Stackebrandt & Goebel, 1994). This probability is supported by the number of phenotypic differences observed between strain Con a/3T and the type strain of the closely related species of B. barbaricus (DSM 14730T) (Table 1), and the type strains of other arsenic-resistant species of Bacillus, namely Bacillus arseniciselenatis (DSM 15340T), Bacillus selenitireducens (DSM 15326T) and B. indicus (Sd/3T). Significant differences in the levels of the fatty acids C13 : 0, iso-C15 : 1, iso-C16 : 0, C16 : 1(7cis), iso-C15 : 0 and anteiso-C15 : 0 further differentiate strain Con a/3T from B. barbaricus (DSM 14730T), when grown under identical conditions (Table 2). Moreover, strain Con a/3T was able to grow in the presence of 20 mM arsenate [As(V)] and 0·3 mM arsenite [As(III)], unlike the above species. Strain Con a/3T also differs from B. indicus Sd/3T, an arsenic-resistant bacterium (Suresh et al., 2004a), in that B. indicus Sd/3T is resistant to higher concentrations of arsenite (3 mM), and the similarity at the 16S rRNA gene level is only 91 %. Therefore, based on the phenotypic, chemotaxonomic and phylogenetic characteristics, strain Con a/3T merits recognition as representing a novel species, for which the name Bacillus arsenicus sp. nov. is proposed.
Description of Bacillus arsenicus sp. nov.
Bacillus arsenicus (ar.sen.i'cus. N.L. masc. adj. arsenicus pertaining to arsenic).
Cells are strictly aerobic, Gram-positive, motile, sporulating and rod-shaped, 1·4–2·0 µm long and 0·3–0·5 µm wide. Endospores form subterminally in non-swelling sporangia. Colonies on nutrient agar are cream, circular, raised, smooth, convex and 1–2 mm in diameter. Optimum growth occurs at 30 °C. Growth occurs at 20–40 °C, but not at 45 °C. Growth occurs at pH 5·5–8·0. Tolerates up to 1·0 % NaCl. Positive for catalase, amylase and protease, but negative for arginine dihydrolase. Hydrolyses starch and gelatin but not urea, Tween 20 or aesculin. Does not reduce nitrate to nitrite and is negative for the methyl red test, indole production, Voges–Proskauer test, citrate utilization and H2S production. Utilizes D-fructose, D-glucose, D-maltose and L-histidine, but not D-cellobiose, L-arabinose, fumarate, glutarate, L-arginine, aspartic acid, creatine or tyrosine as the sole carbon source. Acid is produced from D-glucose and D-maltose, but not from lactose or D-xylose. Strain Con a/3T is sensitive to the antibiotics chloramphenicol (30 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (30 µg), rifampicin (30 µg), streptomycin (10 µg) and tetracycline (30 µg), and is resistant to amoxicillin (10 µg) and ampicillin (10 µg). Other characteristics are listed in Table 1. The major fatty acids are iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C16 : 1(7cis) (Table 2), and the phospholipids present are phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major respiratory quinone is MK-7. The diamino acid found in the cell peptidoglycan is diaminopimelic acid. The G+C content of the DNA is 35·0 mol%.
The type strain is Con a/3T (=MTCC 4380T=DSM 15822T=JCM 12167T).
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ACKNOWLEDGEMENTS |
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