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1 Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22 100 Lund, Sweden
2 Centro de Biotecnología, Facultad de Ciencias y Tecnología, Universidad Mayor de San Simón, Cochabamba, Bolivia
Correspondence
Rajni Hatti-Kaul
Rajni.Hatti-Kaul{at}biotek.lu.se
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of LBB3T is AY376312.
A phylogenetic tree and electron micrographs are available as supplementary material in IJSEM Online.
Present address: PROIMI – CONICET, Av. Belgrano y Pasaje Caseros, 4000 San Miguel de Tucumán, Argentina. 
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; Nielsen et al., 1994, 1995). Soda lakes contain dense populations of aerobic organotrophic and alkaliphilic bacteria. Some of these bacteria are believed to have biotechnological potential as a source of alkali-stable enzymes (Gessesse & Gashe, 1997; Martins et al., 2001).
Strain LBB3T was recovered from liquid samples collected from Lake Bogoria, Kenya in a screening medium for lipolytic micro-organisms (Vargas et al., 2004), composed of 3 % (v/v) olive oil and basal salts (w/v) [0·08 % K2HPO4, 0·06 % KH2PO4, 0·1 % (NH4)2SO4, 0·02 % MgSO4.7H2O, 0·005 % CaCl2.2H2O, 0·3 % NaCl, 0·0001 % FeCl3]. It was grown at 37 °C and pH 10 on an orbital shaker at 200 r.p.m. Cell morphology was examined using a Nikon Optiphot-2 microscope at 1000x magnification. Bacterial size was determined in living cell preparations. Cells were harvested from liquid cultures, washed twice with water and dehydrated through a graded series of ethanol and isopropyl alcohol. Cells were then mounted on 12 mm cover slips, dried in a vacuum desiccator overnight and then gold–palladium (80/20) coated. Biochemical characteristics were screened by using the API 20 E system (bioMérieux) according to Logan & Berkeley (1984). Sugar assimilation was determined by using the API 50 CHB system.
Genomic DNA was extracted and purified according to Marmur (1961), and its purity was assessed from the A260/A280 and A260/A230 ratios (Johnson, 1994). Universal primers were used to amplify 16S rRNA genes (8-27F, AGAGTTTGATCCTGGCTCAG; 1422R, GGTTACCTTGTTACGACTT) (Weisburg et al., 1991). PCR products were purified using the QIA quick PCR Purification kit (Qiagen) and then resuspended in a final volume of 40 µl. DNA sequencing on both strands was performed by using the dideoxy chain-termination method with an ABI Prism 3100 DNA Analyzer, using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems) according to the manufacturer's protocol. GenBank and RDP databases were used to search for 16S rRNA gene sequence similarities (Maidak et al., 2000). A 16S rRNA gene sequence analysis was performed with the aid of the DNAMAN 4.03 software package by using the neighbour-joining method and the Jukes–Cantor distance correction matrix method (Saitou & Nei, 1987). Determination of peptidoglycan type was carried out as described previously (Schleifer & Kandler, 1972; Schleifer, 1985), with the modification that TLC on cellulose was used instead of paper chromatography.
Genomic G+C content (mol%) and DNA–DNA hybridization were determined by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. (1997). G+C content was calculated according to the method of Mesbah et al. (1989). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) with the modifications described by Huss et al. (1983) and Escara & Hutton (1980) using a Gilford System model 2600 spectrophotometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program described by Jahnke (1992).
An almost-complete 16S rRNA gene sequence was determined for strain LBB3T and compared with other available sequences in the GenBank and RDP databases (Fig. A, available as supplementary material in IJSEM Online). This analysis placed the novel strain in the family Bacillaceae, with the closest related micro-organisms being Bacillus pseudofirmus (DSM 8715T) and Bacillus alcalophilus (DSM 485T), bearing 95 and 96 % sequence similarity, respectively.
A comparison of the morphological and taxonomic characteristics of strain LBB3T with those of phylogenetically related species is shown in Table 1. Strain LBB3T produces cream–yellow, circular, convex, smooth, entire, opaque colonies that are 5–6 mm in diameter after 48 h incubation at 37 °C. It is Gram-positive. Microscopic examination showed the cells to be spore-forming and non-motile rods that are 0·3–0·4 µm wide and 2·0–3·5 µm long. In unstained preparations, cells occur singly, in pairs and in irregular clumps (Fig. B, available as supplementary material in IJSEM Online).
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L-orn–D-Asp. This pattern is typical for members of the genus Bacillus and related genera.
As shown in Table 1
, the G+C content of strain LBB3T is 37·5 mol%. This value lies within the range for low G+C content Gram-positive bacteria. DNA–DNA hybridization analysis revealed 39·0 % similarity to B. pseudofirmus DSM 8715T and 55·5 % similarity to B. alcalophilus DSM 485T. These values for hybridization are significantly lower than the recommended threshold value of at least 70 % accepted as the definition of a novel species (Wayne et al., 1987), hence supporting the distinct position of strain LBB3T in the genus Bacillus. Differences in 16S rRNA gene sequence identity of 3 % and the G+C content justify the description of a novel Bacillus species to accommodate strain LBB3T.
Based on the analyses of morphological, physiological and phylogenetic traits, we propose strain LBB3T to be a novel alkaliphilic, halotolerant species of the genus Bacillus, for which the name Bacillus bogoriensis sp. nov. is proposed.
Description of Bacillus bogoriensis sp. nov.
Bacillus bogoriensis (bo.gor.i.en'sis. N.L. adj. bogoriensis pertaining to Lake Bogoria, a soda lake in Kenya).
Cells are rod-shaped (0·3–0·4x2·0–3·5 µm), Gram-positive, spore-forming and non-motile. Catalase-positive and urease-negative. Nitrate is not reduced to nitrite. Strictly aerobic. Optimal growth occurs aerobically at 37 °C (range 10–40 °C), pH 10 (range 8–11) and maximum salt tolerance of 2 M NaCl. Heterotrophic; utilizes rhamnose, sucrose, L-arabinose, D-xylose, fructose, mannose, amygdalin, salicin, cellobiose, maltose, arbutin, aesculin, lactose, trehalose, gentiobiose, starch, glycogen, glycerol, ribose, raffinose, N-acetylglucosamine and 5-ketogluconate. Does not grow on mannitol, erythritol, D-arabinose, L-xylose, adonitol, methyl -xyloside, D-glucose, galactose, melibiose, L-sorbose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, inulin, melezitose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, gluconate or 2-ketogluconate. DNA G+C content is 37·5 mol% (determined by HPLC).
The type strain is LBB3T (=ATCC BAA-922T=LMG 22234T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 655–682. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
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Martins, R. F., Davids, W., Abu Al-Soud, W., Levander, F., Rådström, P. & Hatti-Kaul, R. (2001). Starch-hydrolyzing bacteria from Ethiopian soda lakes. Extremophiles 5, 135–144.[CrossRef][Medline]
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Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141, 1745–1761.
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