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Int J Syst Evol Microbiol 57 (2007), 238-242; DOI  10.1099/ijs.0.64617-0
© 2007 International Union of Microbiological Societies

Bacillus lehensis sp. nov., an alkalitolerant bacterium isolated from soil

A. Ghosh1, M. Bhardwaj2, T. Satyanarayana2, M. Khurana1, S. Mayilraj1 and R. K. Jain1

1 Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector-39A, Chandigarh 160036, India
2 Microbiology, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110021, India

Correspondence
R. K. Jain
rkj{at}imtech.res.in


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A Gram-positive, endospore-forming, alkalitolerant bacterial strain, designated MLB2T, was isolated from soil from Leh, India, and was subjected to a polyphasic taxonomic study. The strain exhibited phenotypic properties that included chemotaxonomic characteristics consistent with its classification in the genus Bacillus. Growth was observed at pH 7.0–11.0, but not at pH 6.0. The DNA G+C content was 41.4 mol%. The highest level of 16S rRNA gene sequence similarity was with Bacillus oshimensis JCM 12663T (98.8 %). However, DNA–DNA hybridization experiments indicated low levels of genomic relatedness with the type strains of B. oshimensis (62 %), Bacillus patagoniensis (55 %), Bacillus clausii (51 %) and Bacillus gibsonii (34 %), the species with which strain MLB2T formed a coherent cluster (based on the results of the phylogenetic analysis). On the basis of the phenotypic characteristics and genotypic distinctiveness of strain MLB2T, it should be classified within a novel species of Bacillus, for which the name Bacillus lehensis sp. nov. is proposed. The type strain is MLB2T (=MTCC 7633T=JCM 13820T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MLB2T is AY793550.

A table showing the fatty acid compositions of strain MLB2T and its close relatives is available as supplementary material in IJSEM Online.


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In recent years there has been increasing interest in alkaliphilic and alkalitolerant micro-organisms, attributable to their ability to grow under extreme conditions as well as to the use of their enzymes in biotechnological applications. Naturally occurring alkaline environments, which include soda lakes, deserts and arid soils, harbour a wide range of alkaliphilic and alkalitolerant micro-organisms (Ulukanli & Diurak, 2002Go). The genus Bacillus currently includes 19 species characterized as alkaliphilic and alkalitolerant (http://www.bacterio.cict.fr/b/bacillus.html), and many of them have been studied with a view to finding industrial applications (Horikoshi, 1991Go; Nielsen et al., 1995Go; Yumoto et al., 1998Go). Alkaliphilic enzymes (such as alkaline cellulases and alkaline proteases) produced by Bacillus species are used in the detergent industries (Horikoshi, 1999Go). In this work, we describe an alkalitolerant, endospore-forming bacterium, designated MLB2T, isolated from pristine soil samples from Leh, India (at 3500 m above sea level and at temperatures ranging from –20 to 30 °C). Characterization of the protease produced by the strain indicated optimal activity at pH 10.0.

Strain MLB2T was isolated on tryptone soya agar (TSA, pH 7.3; HiMedia) at 25 °C by means of the dilution plating technique. Subculturing was performed on TSA at 25 °C for 24 h and the bacterial isolate was maintained as glycerol stock at –70 °C. Reference strains used were Bacillus oshimensis JCM 12663T (=MTCC 7915T), obtained from the Japan Collection of Microorganisms (Saitama, Japan), and Bacillus patagoniensis DSM 16117T (=MTCC 7916T), Bacillus clausii DSM 8716T (=MTCC 7914T), Bacillus gibsonii DSM 8722T (=MTCC 7917T) and Bacillus alcalophilus DSM 485T (=MTCC 7913T), from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany).

Colony morphology was examined by studying the growth of the strain on TSA at 25 °C for 24 h. Cell morphology was investigated by means of light microscopy (Zeiss) at x1000 and scanning electron microscopy (Stereoscan 260; Leica). Motility was checked using the method described by Skerman (1967)Go. The Gram reaction was determined using the HiMedia Gram-staining kit according to the manufacturer's instructions. Growth at different temperatures and NaCl concentrations was studied as described by Cowan & Steel (1965)Go. Growth at different pH values was tested using tryptone soya broth (HiMedia) as growth medium and the pH was adjusted using appropriate biological buffers (Nakajima et al., 2005Go). All biochemical and physiological studies were carried out at pH 8.0. The following characteristics were determined as described by Cowan & Steel (1965)Go: hydrolysis of gelatin, casein, starch, hippurate, ONPG and 4-methylumbelliferone glucuronide; Voges–Proskauer, methyl red, oxidation–fermentation tests; catalase and oxidase (oxidation of tetramethyl-p-phenylenediamine dihydrochloride; Sigma) activities; growth on Simmons' citrate and MacConkey agar; production of H2S and indole; reduction of nitrate; tyrosinase activity; arginine hydrolysis; and deamination of phenylalanine. The assimilation of various substrates for growth was determined by using a Biolog GP2 MicroPlate as described by Mayilraj et al. (2006)Go. Acid production from various carbohydrates was tested as described by Claus & Berkeley (1986)Go. The sensitivity of the strain to various antibiotics was tested using antibiotic-susceptibility discs (HiMedia).

Freeze-dried cells used for chemotaxonomic analyses (with the exception of the fatty acid study) were prepared after growth of the strain in tryptone soya broth for 24 h at 25 °C. Polar lipids were analysed as described by Suresh et al. (2004)Go. The diagnostic amino acids and whole-cell sugars were determined using TLC, as described by Staneck & Roberts (1974)Go. Isoprenoid quinones were extracted (Minnikin et al., 1984Go) and separated by reversed-phase HPLC (Kroppenstedt, 1982Go). For cellular fatty acid analysis, the strains were grown on TSA and a fatty acid methyl ester analysis was then performed with the Sherlock Microbial Identification System (MIDI), as described previously (Pandey et al., 2002Go). The G+C content of genomic DNA was determined spectrophotometrically (Lambda 35; Perkin Elmer) using the thermal denaturation method (Mandel & Marmur, 1968Go).

The genomic DNA was isolated using a genomic DNA-isolation kit (Qiagen). PCR amplification, cloning and sequencing of the 16S rRNA gene were performed as described previously (Ghosh et al., 2006Go). The 16S rRNA gene sequences of closely related taxa with validly published names were retrieved from the GenBank database using BLASTN (Altschul et al., 1997Go) and aligned using the CLUSTAL_X program (Thompson et al., 1997Go); the alignment was edited manually. For the neighbour-joining analysis (Saitou & Nei, 1987Go), the distances between the sequences were calculated using the method of Jukes & Cantor (1969)Go. A bootstrap analysis of 1000 replications was performed to assess the confidence limits of the branching (Felsenstein, 1985Go). DNA–DNA hybridization was performed each time with freshly isolated genomic DNA and was repeated three times using the membrane filter method (Tourova & Antonov, 1987Go).

The phenotypic properties are shown in detail in Table 1Go and also in the species description. Growth was observed at pH 7.0–11.0, but not at pH 6.0, and the optimum pH for growth (pH 8.0) confirmed strain MLB2T to be an alkalitolerant bacterium. The cells of strain MLB2T were sensitive to the following antibiotics (µg per disc): ampicillin (10), chloramphenicol (30), ciprofloxacin (5), erythromycin (10), furazolidone (50), gentamicin (10), nalidixic acid (30), neomycin (30), oleandomycin (15), rifampicin (5), spectinomycin (100), streptomycin (10), sulphadiazine (300), sulphamethizole (300) and tetracycline (30). The cells were resistant to the following antibiotics (µg per disc): cephaloridine (30), cloxacillin (5), colistin (10), novobiocin (30), penicillin (1.5), sulphafurazole (100), tobramycin (10) and trimethoprim (25).


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  		Table 1. Characteristics that differentiate strain MLB2T from phylogenetically related type strains

Strains: 1, MLB2T; 2, B. oshimensis JCM 12663T; 3, B. patagoniensis DSM 16117T; 4, B. clausii DSM 8716T; 5, B. gibsonii DSM 8722T; 6, B. alcalophilus DSM 485T. All strains were found to produce acid from glycerol, 2-ketogluconate and D-maltose but not from inositol or D-melezitose. All strains were positive for the assimilation of acetic acid, D-fructose, D-maltose and D-mannose, but negative for the assimilation of adenosine, D-cellobiose, 2-deoxyadenosine, D-fructose 6-phosphate, L-fucose, D-glucose 6-phosphate, D-gluconic acid, DL-{alpha}-glycerol phosphate, beta-hydroxybutyric acid, inosine, {alpha}-ketoglutaric acid, methyl {alpha}-D-galactoside, methyl {alpha}-D-glucoside, stachyose and succinic acid as growth substrates (Biolog). All data were obtained from this study unless otherwise indicated. +, Positive; W, weakly positive; –, negative.

 
Most of the chemotaxonomic properties (presented in the species description) were typical of members of the genus Bacillus. The isoprenoid quinones present in strain MLB2T were MK-7 and MK-6. The fatty acid methyl ester profile of the novel strain matched qualitatively those of phylogenetically related species (see Supplementary Table S1 available in IJSEM Online). The DNA G+C content was calculated as 41.4 mol% (mean of three replications), which falls within the defined range (32–69 mol%) accepted for Bacillus species (Fritze et al., 1990Go).

The complete sequence (1541 bases) of the 16S rRNA gene of strain MLB2T was determined and was compared with those of closely related taxa retrieved from the GenBank database. The phylogenetic tree constructed using the neighbour-joining method suggested that this strain is a member of group 6 (Nielsen et al., 1994Go) of the genus Bacillus; MLB2T formed a clade with B. oshimensis K11T with a bootstrap value of 100 % (Fig. 1Go). Pairwise sequence analysis revealed that the highest sequence similarity was with B. oshimensis K11T (98.8 %), followed by B. patagoniensis PAT 05T (98.5 %) and B. clausii DSM 8716T (97.3 %); the remaining species with validly published names showed less than 97 % similarity.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree indicating the position of strain MLB2T among related species of the genus Bacillus. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at the branch points. Staphylococcus aureus ATCC 29740T was used as an outgroup. Bar, 0.1 substitutions per site.

 
In the DNA–DNA hybridization analysis, strain MLB2T exhibited 62±2 % similarity to B. oshimensis JCM 12663T, 55±3 % similarity to B. patagoniensis DSM 16117T, 51±3 % similarity to B. clausii DSM 8716T, 34±3 % similarity to B. gibsonii DSM 8722T and 21±2 % similarity to B. alcalophilus DSM 485T. These values for hybridization were lower than the recommended threshold value accepted for defining a novel species (Wayne et al., 1987Go; Stackebrandt & Goebel, 1994Go), hence supporting the distinct position of strain MLB2T within the genus Bacillus. On the basis of the differential phenotypic (Table 1Go) and genotypic properties, strain MLB2T should be assigned to a novel species of the genus Bacillus, for which the name Bacillus lehensis sp. nov. is proposed.

Description of Bacillus lehensis sp. nov.
Bacillus lehensis (le.hen'sis. N.L. masc. adj. lehensis pertaining to Leh, in India, where the type strain was isolated).

Cells are aerobic, Gram-positive, motile rods (0.5–0.8x1.0–3.8 µm). Oval spores develop subterminally in the cells and sporangia are not swollen. Colonies are circular, convex, smooth and pigmented creamish-yellow. Catalase and oxidase are produced. Negative for H2S, indole and urease production, in the methyl red and Voges–Proskauer tests, for ONPG hydrolysis and in the oxidation–fermentation test. No growth occurs on MacConkey agar or Simmons' citrate agar. Growth occurs at temperatures in the range 10–37 °C (optimum temperature, 25 °C), pH 7.0–11.0 (optimum, pH 8.0) and at NaCl concentrations up to 12 %. Nitrate is reduced to nitrite. Assimilation of substrates for growth and production of acid from carbohydrates under aerobic conditions are shown in Table 1Go. Hydrolyses casein, gelatin, hippurate and starch. Negative for arginine dihydrolase, phenylalanine deaminase and tyrosinase activity; positive for 4-methylumbelliferone glucuronide activity. Cell wall contains meso-diaminopimelic acid as the diagnostic diamino acid; D-glucose, D-galactose and D-xylose are major cell-wall sugars. The predominant polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, along with two unknown phospholipids (PL1 and PL2). The major isoprenoid quinone is MK-7. The major cellular fatty acids are iso-C15 : 0 (57.0 %), anteiso-C15 : 0 (17.5 %) and iso-C17 : 0 (8.2 %). The DNA G+C content is 41.4 mol%.

The type strain, MLB2T (=MTCC 7633T=JCM 13820T), was isolated from soil collected from Leh, India.


   ACKNOWLEDGEMENTS
 
We are indebted to Dr J. P. Euzéby (École Nationale Vétérinaire, France) for his suggestion regarding Latin nomenclature for the novel species. We wish to express our gratitude to Ms Gabi Pötter (DSMZ) for her excellent technical assistance. We also thank Dr K. Suresh for helpful discussions. This work was supported, in part, by CSIR and DBT, Government of India. A. G. acknowledges a research fellowship awarded by CSIR. This is IMTECH communication number 029/2006.


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