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1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
2 Department of Environmental and Information Science, Otsuma Women's University, Tamashi, Tokyo 206, Japan
Correspondence
S. Shivaji
shivas{at}ccmb.res.in
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The EMBL/GenBank/DDBJ accession number for the 16S rRNA gene sequence of strain CMS 21wT is AJ514408.
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to accommodate bacteria that have spherical or oval-shaped cells, low DNA G+C content (40–42 mol%) and MK-7 as the major menaquinone. Sporosarcina species can be differentiated from other members of the Bacillaceae by their coccoid or rod-shaped cells, motility, sporulation and possession of MK-7 as the major menaquinone and A4
as the peptidoglycan variant. The genus Sporosarcina originally comprised two species, Sporosarcina ureae and Sporosarcina halophila (Claus et al., 1983; Claus & Fahmy, 1986). However, three species of the genus Bacillus, namely Bacillus globisporus (Larkin & Stokes, 1967), Bacillus psychrophilus (Nakamura, 1984) and Bacillus pasteurii (Miguel, 1889), which belonged to rRNA group 2 (Ash et al., 1991) and contain L-lysine in their cell wall, have recently been transferred to the genus Sporosarcina (Yoon et al., 2001) as Sporosarcina globispora, Sporosarcina psychrophila and Sporosarcina pasteurii. Yoon et al. (2001) also identified a novel species, Sporosarcina aquimarina. In continuation of our work on the heterotrophic bacteria associated with cyanobacterial mat samples from the lakes of Antarctica (Reddy et al., 2000, 2002a, 2002b, 2003), seven psychrophilic bacteria from a pond in McMurdo Region, Antarctica, have been identified as strains of Sporosarcina.
Source of the organisms, media and growth conditions
Seven pure cultures (CMS 13, CMS 14, CMS 15, CMS 21wT, CMS 89, CMS 91 and CMS 93) were isolated from cyanobacterial mat samples collected from ponds L4, L8 and E4 of Wright Valley, McMurdo Region, Antarctica (Matsumoto, 1993) according to a previously described procedure (Reddy et al., 2000) using ABM agar (0·5 % peptone, 0·2 % yeast extract and 1·5 % agar, pH 7·2; Shivaji et al., 1989). Optimum growth conditions were also determined using ABM agar plates.
All seven isolates were identical with respect to their colony and cell morphologies, growth requirements, salt tolerance and many biochemical characteristics, suggesting that they are probably strains of the same species. In fact, all seven had identical 16S rRNA gene sequences, implying that the seven isolates are clonal in origin. CMS 21wT was chosen as a representative strain for detailed studies to establish its identity.
Morphology, motility and biochemical and chemotaxonomic characteristics
The morphology and motility of CMS 21wT were determined by taking exponential-phase cells and observing them by phase-contrast microscopy (1000x). Biochemical tests were performed on cultures grown at 20 °C in nutrient agar (0·5 % peptone, 0·3 % beef extract, 0·8 % NaCl, pH 7·2). Catalase, oxidase, phosphatase, gelatinase, urease, lipase, arginine dihydrolase and 
-galactosidase activities were determined according to standard methods (Holding & Collee, 1971). Production of indole, methyl red and Voges–Proskauer tests, reduction of nitrate to nitrite, hydrolysis of starch and aesculin and production of acid and gas were assessed according to standard procedures (Hugh & Leifson, 1953; Stanier et al., 1966; Holding & Collee, 1971; Stolp & Gadkari, 1981). Antibiotic sensitivity and growth characteristics were analysed using nutrient agar plates and utilization of carbon compounds was determined on minimal medium. The DNA G+C content was determined as described previously (Shivaji et al., 1991).
Fatty acid methyl esters (Sato & Murata, 1988) were analysed as described previously (Reddy et al., 2002a). Isoprenoid quinones were extracted according to the method described by Collins et al. (1977) and separated by HPLC using an SB-C18 Zorbax reverse-phase column (4·6x250 mm) fixed to a Hewlett-Packard series 1100 HPLC connected to a Hewlett-Packard programmable fluorescence detector (Tamaoka et al., 1982). An isocratic gradient of methanol/isopropyl ether (3 : 1, v/v) was used for elution; the flow rate and filter used were respectively 0·5 ml min-1 and 270 nm. Peptidoglycan was prepared and analysed according to the method described by Komagata & Suzuki (1987).
Phenotypic and chemotaxonomic characteristics of CMS 21wT are described in the species description. From these characteristics, it is apparent that CMS 21wT, which is rod-shaped, non-motile and Gram-positive with subterminal spores, is phenotypically similar to species of the genus Sporosarcina. This was confirmed by chemotaxonomic characteristics such as A4 peptidoglycan variant (with L-lys–D-Glu peptidoglycan type), MK-7 as the major menaquinone, anteiso-C15 : 0 as the major fatty acid and a DNA G+C content of 44 mol%. The phenotypic and chemotaxonomic characteristics that differentiate CMS 21wT from the five reported type strains of Sporosarcina and Bacillus insolitus are listed in Table 1.
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). The partial sequence of the 16S rRNA gene containing 1488 bp was aligned with closely related sequences using CLUSTAL W (Thompson et al., 1994). Pairwise evolutionary distances were computed using the DNADIST program with Kimura's two-parameter model (Kimura, 1980) and phylogenetic trees were constructed using four tree-making algorithms (UPGMA, KITCH, FITCH and DNAPARS) (Felsenstein, 1993). Phylogenetic trees were also made using DNADIST, UPGMA and CONSENSE after resampling the data 1000 times using SEQBOOT and bootstrap analysis (Felsenstein, 1993).
BLAST sequence similarity analysis using the 16S rRNA gene sequence indicated that CMS 21wT is closely related to Sporosarcina species. This was further confirmed by phylogenetic analysis of the 1488 bp 16S rRNA gene sequence of CMS 21wT and sequences from species of the genera Sporosarcina, Bacillus, Caryophanon, Kurthia and Planococcus and by constructing a UPGMA-based phylogenetic tree. The evolutionary distance calculated using Kimura's two-parameter model indicated that CMS 21wT is related to species of the genus Sporosarcina (similarity of 93–96 %) and is most closely related to S. globispora (96 %), S. psychrophila (96 %) and B. insolitus (96 %). The topology of the phylogenetic tree showed that CMS 21wT is closely related to S. globispora (Rüger, 1983), S. psychrophila (Nakamura, 1984), S. aquimarina (Yoon et al., 2001), S. ureae (Claus & Fahmy, 1986), S. pasteurii (Yoon et al., 2001) and B. insolitus (Rüger, 1983). However, within the cluster, only S. psychrophila and S. globispora had a high bootstrap value (100 %); this value varied from 40 to 60 % with the other species (Fig. 1). All the different algorithms used for tree construction gave comparable branching patterns. However, CMS 21wT cannot be grouped with B. insolitus since the latter strain has ornithine in its peptidoglycan, unlike all reported species of Sporosarcina, which contain lysine (Yoon et al., 2001). Furthermore, inclusion of B. insolitus in the cluster is not very robust, having a low bootstrap value of 55 % with strain CMS 21wT.
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). Therefore, it would appear that CMS 21wT represents a novel species of Sporosarcina. Further evidence that CMS 21wT represents a novel species comes from differences in phenotypic characteristics (Table 1
). CMS 21wT is unique in that it has both anteiso-C15 : 0 and iso-C16 : 1 as major fatty acids, whereas other species of the genus Sporosarcina have only antesio-C15 : 0 as the major fatty acid (Yoon et al., 2001). CMS 21wT also contains C15 : 1, which is absent in other species of the genus Sporosarcina. Furthermore, in B. insolitus, the major fatty acids are iso-C15 : 0, anteiso-C15 : 0 and C16 : 1 (Kaneda et al., 1983). Therefore, CMS 21wT is proposed as the type strain of a novel species of the genus Sporosarcina, Sporosarcina macmurdoensis sp. nov.
Description of Sporosarcina macmurdoensis sp. nov.
Sporosarcina macmurdoensis (mac.mur.do.en'sis. N.L. fem. adj. macmurdoensis pertaining to the McMurdo Region, Antarctica, where the isolates were collected).
Colonies are circular, flat, irregular, opaque and white with a diameter of 2–3 mm. Cells are rod-shaped, single and non-motile and form subterminal spores. Cells can tolerate a maximum of 3 % (w/v) NaCl and grow at pH 6–9 (pH 7 is optimum for growth). Psychrophilic; grows at 4–25 °C (optimum growth temperature 18–20 °C). Positive for catalase, phosphatase, gelatinase and starch hydrolysis and negative for lipase, urease, oxidase, -galactosidase, arginine dihydrolase, arginine decarboxylase, lysine decarboxylase, indole production, methyl red test, Voges–Proskauer test, aesculin hydrolysis and nitrate reduction to nitrite. Does not produce acid or gas from L-arabinose, D-fructose, D-galactose, lactose, D-mannose, D-mannitol, L-rhamnose, sucrose or D-xylose. Utilizes dulcitol, D-fructose, D-galactose, D-glucose, meso-inositol, lactose, D-maltose, D-mannose, pyruvate, D-raffinose, D-xylose and L-glutamic acid as sole carbon sources, but not acetate, adonitol, L-arabinose, D-cellobiose, cellulose, citrate, dextran, glucose, meso-erythritol, fumaric acid, glycerol, inulin, lactic acid, D-mannitol, D-melibiose, melezitose, L-rhamnose, D-ribose, sorbitol, D-sorbose, sucrose, succinic acid, trehalose, thioglycollate, L-alanine, L-arginine, L-aspartic acid, L-aspargine, L-glutamine, L-lysine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-tryptophan or L-valine. Sensitive to amikacin, ampicillin, amoxycillin, bacitracin, carbenicillin, cefazoline, cefaperazone, cephotaxime, chloramphenicol, chlorotetracycline, co-trimoxazole, ciprofloxacin, erythromycin, furazolidone, furoxone, gentamicin, kanamycin, lomefloxacin, nalidixic acid, neomycin, nitrofurazone, nitrofurantoin, norfloxacin, novobiocin, nystatin, oxytetracycline, penicillin, polymyxin-B, rifampicin, roxithromycin, streptomycin, tetracycline, tobramycin, trimethoprim and vancomycin, but resistant to cefuroxime, colistin and lincomycin. Peptidoglycan type is L-lys–D-Glu of the A4 variant and MK-7 is the major menaquinone. When grown in nutrient broth at 22 °C, the fatty acid composition is as follows (proportions of total fatty acids in parentheses): C13 : 0 (0·5 %), C14 : 0 (0·5 %), iso-C14 : 0 (11·1 %), C15 : 0 (1·1 %), iso-C15 : 0 (4·0 %), anteiso-C15 : 0 (37·4 %), C15 : 1 (1·5 %), C16 : 0 (0·7 %), iso-C16 : 0 (3·2 %), C16 : 1
7c (1·1 %), iso-C16 : 1 (22·6 %), iso-C17 : 0 (3·0 %) and anteiso-C17 : 0 (13·0 %).
The DNA G+C content of the type strain, CMS 21wT (=MTCC 4670T=DSM 15428T=CIP 107784T), is 44 mol%.
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ACKNOWLEDGEMENTS |
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