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1 Microbial Gene Research and Resources Facility, School of Biomolecular and Biomedical Sciences, Griffith University, Brisbane, Australia 4111
2 Department of Biosciences, Saurashtra University, Rajkot, India 360005
Correspondence
B. K. C. Patel
b.patel{at}griffith.edu.au
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) and have attracted much attention as they secrete extracellular enzymes that are both active and stable at high salt concentrations and high pH. These properties make them an interesting tool for fundamental research and for biotechnological exploration (Margesin & Schinner, 2001; Demirjian et al., 2001). The vast majority of haloalkaliphiles and alkalitolerant species have been isolated from athalassohaline environments (Duckworth et al., 1996; Jones et al., 1998; Pikuta et al., 2003; Rees et al., 2004) while thalassohaline environments have remained relatively less explored (Neilsen et al., 1995; Suzuki et al., 1997; Munoz et al., 2001; Amoozegar et al., 2003). We describe in this report the characterization of a new halotolerant and alkalitolerant Bacillus species that was isolated from a thalassohaline environment. A soil sample was collected from a natural saltpan near the shore of Port Okha (22° 15' N, 69° 1' E) in sterile glass containers by scraping the soil surface. For initiating enrichment cultures, 1 g soil was diluted in 5 ml sterile distilled H2O, and large suspended solids were allowed to settle by gravitation for 10 min, after which 1 ml of the relatively clear suspension was added to a flask containing 100 ml complex medium (CMB) consisting of (g l–1): glucose, 10; peptone, 5; yeast extract, 5; KH2PO4, 5; NaCl, 10–25 %. The medium was adjusted to pH 10.0 using sterile autoclaved Na2CO3 (20 %, w/v). CMB medium became turbid after 48 h incubation at 37 °C and pure cultures were isolated by streaking CMB agar plates (CMB medium containing 2.5 % agar). Single colonies that developed after 4 days incubation at 37 °C on CMB plates were restreaked onto fresh CMB agar plates. The isolation procedure was repeated three times to obtain the pure culture. One of the pure cultures, Kh10-101T, was designated a type strain, and characterized further. The pure culture was maintained on CMB medium (pH 9; 10 %, w/v, NaCl) described above and stored at 4 °C. The strain was subcultured at monthly intervals. Colonies of Kh10-101T were round, flat and translucent with irregular margins, a smooth surface and a creamish pigment.
Cellular characterization and sporulation tests performed as described previously (Kanso & Patel, 2003) showed that the cells of strain Kh10-101T were rod-shaped (0.6–0.8x2–3 µm) and actively motile. Electron microscopy of negatively stained cells showed the presence of up to three subpolar flagella. The cells stained Gram-positive and thin sections revealed the presence of a typical thick Gram-positive cell wall. Microscopic examination did not reveal the presence of inclusion bodies or spores. The cells of strain Kh10-101T were not viable after 10 min incubation at 70 °C, indicating the absence of heat-resistant bodies such as spores.
The optimal conditions for growth of strain Kh10-101T were tested in a medium containing (per 100 ml double-distilled H2O): 1 g D-glucose, 0.5 g KH2PO4, 0.5 g peptone, 0.5 g yeast extract and 5 g NaCl. The strain grew optimally at pH 9 (pH growth range pH 7–10) with 5 % NaCl (NaCl growth range 0–10 %) at 37 °C (temperature growth range 25–40 °C). The generation time was determined to be 2.5 h under optimal growth conditions (CMB medium, pH 9, containing 5 % NaCl and incubation temperature of 37 °C).
To determine antibiotic sensitivity, an active culture of strain Kh10-101T was inoculated onto CMB agar plates (pH 9) containing 10 % (w/v) NaCl following which Hi-media octadiscs impregnated with antibiotics were placed on their surface. The plates were incubated at 37 °C for up to 48 h and the zones of inhibition around the antibiotic discs were recorded. Strain Kh10-101T was found to be highly sensitive to lincomycin, meticillin, cefuroxime and cephalexin but resistant to gentamicin, tetracycline and cotrimazine.
Biolog GP2 plates (Hayward) were used to characterize the biochemical capacity of the strain using the manufacturer's recommended procedures but with the following modification. A culture of strain Kh10-101T was centrifuged and the cell pellet was washed and resuspended in a tube of Inoculating Fluid (IF GN/GP) supplied with the Biolog plates, to which was added 1.25 g NaCl and 1.2 g Na2CO3. The resulting fluid was filter-sterilized prior to use. Cell suspension (150 µl) was then added to the wells of Biolog GP2 plates, and the plates were incubated at 37 °C and read after 24 h as recommended by the manufacturer. Strain Kh10-101T could metabolize 3-methyl glucose, D-fructose, D-fructose 6-phosphate, D-galactose, D-glucose, D-galacturonic acid, D-mannose, D-psicose, D-ribose, D-tagatose, D-xylose, gentiobiose, lactulose, L-arabinose, L-fucose, L-rhamnose, palatinose and turanose. CMB agar plates lacking glucose but containing casein (2.5 %), gelatin (0.004 %) or starch (0.01 %) were used to test for caseinase, gelatinase and amylase activities, respectively, using the methods described by Barrow & Feltham (1993). Strain Kh10-101T was capable of hydrolysing casein, gelatin and starch. Amylase production occurred at pH 7–10 and 0–10 % (w/v) NaCl at an optimum of 5 % NaCl and pH 9.
Catalase activity was positive as determined by dropping H2O2 on to colonies that developed on CMB agar plates. Aromatic compounds (p-methylhydroxybenzoate, p-ethylhydroxybenzoate, p-propylbenzoate and sodium benzoate) tested as carbon sources (0.03 %) in a medium (pH 9.0) containing (per 100 ml) 0.05 g peptone, 0.05 g yeast extract, 1 g KH2PO4 and 5 g NaCl were found not to be used. No growth was observed with Tween 40 or Tween 80.
Genomic DNA was prepared using a modified method (Marmur & Doty, 1961) in which achromopeptidase was added at a final concentration of 1 mg ml–1 for cell lysis and RNase was added at a final concentration of 20 µg ml–1 to digest RNA (Kanso & Patel, 2003). The DNA was dissolved overnight at 4 °C in 0.1x SSC to a concentration of 20 µg ml–1. Escherichia coli genomic DNA prepared in the same manner was used as reference DNA. The DNA G+C content of strain Kh10-101T was 41±1 mol%, determined by the thermal denaturation temperature (Tm) using a Cintra20 spectrophotometer (GBC Scientific Equipment).
The methods used for 16S rRNA gene amplification and sequencing have been reported previously (Andrews & Patel, 1996). Partial sequences generated in this investigation were assembled and the consensus sequence was corrected manually for errors using BioEdit version 5.0.1 (Hall, 1999). The most closely related sequences against GenBank and Ribosomal Database Project II were identified using BLAST (Altschul et al., 1997) and the Sequence Match program (Maidak et al., 2001), extracted, aligned and manually adjusted according to the 16S rRNA secondary structure using BioEdit. Sequence uncertainties were omitted and phylogenetic reconstruction was achieved using TreeCon (Van de Peer & De Wachter, 1994) in which pairwise evolutionary distances were computed from percentage similarities (Jukes & Cantor, 1969) and phylogenetic trees were constructed from the evolutionary distances using the neighbour-joining method (Saitou & Nei, 1987). Tree topology was re-examined by the bootstrap method of resampling (Felsenstein, 1985) using 1000 bootstraps. BLAST results indicated a 96 % similarity to the sixth 16S rRNA group represented by the cluster of haloalkaliphilic and alkalitolerant members of the genus Bacillus and other related bacteria with the closest relative being Bacillus krulwichiae (96 % similarity) (Fig. 1). In addition to the phylogenetic distance separating strain Kh10-101T and B. krulwichiae, a great number of phenotypic differences also suggest that strain Kh10-101T is a different species (Table 1). For example, strain Kh10-101T did not utilize Tween 40, Tween 80, aromatic compounds or a range of carbohydrates (mannitol, trehalose and sucrose) but B. krulwichiae did. Also, strain Kh10-101T used lactose, L-rhamnose and D-mannose but B. krulwichiae did not. Strain Kh10-101T was a halotolerant alkaliphile and grew at neutral pH and with NaCl concentrations of up to 10 % whereas B. krulwichiae was a halotolerant obligate alkaliphile which grew with NaCl concentrations of up to 14 %.
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Description of Bacillus okhensis sp. nov.
Bacillus okhensis (ok.hen'sis. N.L. masc. adj. okhensis pertaining to Port Okha, a port of the Dwarka region in India, where the type strain was isolated).
Cells are Gram-positive rods (0.6–0.8x2–3 µm) with a subterminal flagella tuft allowing vigorous forward propulsion. Spores are not observed and cells are sensitive to heat. Colonies are round and creamish with irregular edges. Growth occurs at pH 7–10 and 0–10 % (w/v) NaCl. Grows optimally with 5 % NaCl (NaCl growth range 0–10 %). Sensitive to lincomycin, meticillin, cefuroxime and cephalexin but resistant to gentamicin, tetracycline and cotrimazine. Utilizes 3-methyl glucose, D-fructose, D-fructose 6-phosphate, D-galactose, D-glucose, D-galacturonic acid, D-mannose, D-psicose, D-ribose, D-tagatose, D-xylose, gentiobiose, lactulose, L-arabinose, L-fucose, L-rhamnose, palatinose and turanose but not Tween 40 or 80, glycerol, mannitol, sucrose, adenosine or dextrin. Hydrolyses casein, starch and gelatin, produces catalase and can not reduce nitrate. The DNA G+C content is 41.6±1 mol%.
The type strain, Kh10-101T (=JCM 13040T=ATCC BAA-1137T), was isolated from an Indian saltpan.
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