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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
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Sisinthy Shivaji
shivas{at}ccmb.res.in
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Published online ahead of print on 28 June 2002 as DOI 10·1099/ijs.0·02336-0.
The EMBL accession number for the 16S rDNA sequence of Kocuria polaris CMS 76orT is AJ278868.
Pigment characteristics of strain CMS 76orT are available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
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). At the time of writing, the genus Kocuria contained five species, i.e. Kocuria rosea, Kocuria varians, Kocuria kristinae, Kocuria palustris and Kocuria rhizophila. All of the recognized species of Kocuria are coccoid, Gram-positive, non-endospore-forming, aerobic, non-halophilic micro-organisms that can be differentiated from other genera in the order Actinomycetales on the basis of their peptidoglycan type (L-Lys–Ala3/4), the presence of galactosamine and glucosamine as their major cell-wall amino sugars, the presence of MK-7(H2) and MK-8(H2) as their major menaquinones, the presence of diphosphatidylglycerol and phosphatidylglycerol, the presence of the fatty acid anteiso-C15 : 0 and the G+C content of their DNA, which ranges from 66 to 75 mol%. In addition, all of the reported species have been observed to be mesophilic (Stackebrandt et al., 1995). In the present study, a psychrophilic bacterium (strain CMS 76orT) isolated from a cyanobacterial mat sample originating from a pond in Antarctica was identified as a novel species of the genus Kocuria on the basis of its morphological, chemotaxonomic and phylogenetic properties. We propose the name Kocuria polaris for this novel species.
Strain CMS 76orT was isolated from a cyanobacterial mat sample (predominantly containing Phormidium sp.) collected from pond L9 (77°33'3''S, 160°38'E) located in Wright Valley, McMurdo, Antarctica (Matsumoto, 1993). The mat sample (200 mg) was processed, plated onto Antarctic bacterial medium (ABM) plates containing 0·5 % (w/v) peptone, 0·2 % (w/v) yeast extract and 1·5 % (w/v) agar (pH 6·9) and incubated at 5 °C (Shivaji et al., 1992) for 15 days. The colonies that appeared were predominantly white in colour, except those of strains CMS 76orT and CMS 90rT, which were orange (‘or’ suffix) and red (‘r’ suffix), respectively. Strain CMS 90rT has subsequently been described as Arthrobacter roseus (Reddy et al., 2002a).
The shape and motility of cultures from the lag, exponential and stationary phases of growth were observed by phase-contrast microscopy (magnification, x1000). Motility was determined by the hanging drop method, and staining of the flagellum was done by the silver impregnation method (Blenden & Goldberg, 1965). All enzyme tests were performed by growing strains at 20 °C in the appropriate medium according to standard methods (Holding & Collee, 1971). The production of indole, the methyl red test, the Voges–Proskauer test, levan formation, reduction of nitrate to nitrite, hydrolysis of starch and aesculin, and the formation of acid and gas were also monitored according to standard methods (Hugh & Leifson, 1953; Stanier et al., 1966; Holding & Collee, 1971; Stolp & Gadkari, 1981). The abilities of strain CMS 76orT, K. rosea ATCC 186T and K. rosea ATCC 187 (formerly the type strain of Kocuria erythromyxa) to utilize various carbon compounds as sole carbon sources were tested using minimal medium [1·05 % K2HPO4, 0·45 % KH2PO4, 0·1 % (NH4)2SO4, 0·5 % carbon source]. Determinations of sensitivity to different antibiotics and of the optimal temperature, pH and salt values for growth were done on ABM plates. K. rosea ATCC 186T, K. rosea ATCC 187 and Sphingobacterium antarcticum ATCC 51969T were used as controls in this study.
The phenotypic properties of strain CMS 76orT are given in the species description for K. polaris. Characteristics useful for distinguishing strain CMS 76orT from its closest relatives are listed in Tables 1 and 2.
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). The almost-complete 16S rDNA sequence of strain CMS 76orT (1451 nt) was aligned with 14 reference sequences deposited in EMBL, using CLUSTAL V (Higgins et al., 1992). Furthermore, following an initial crude taxonomic affiliation, a more-detailed analysis was done using the 16S rDNA sequences of members of the genera Kocuria, Micrococcus, Nesterenkonia, Dermacoccus and Renibacterium. The DNADIST program with the Kimura-2 correction model (Kimura, 1980) was used to compute the pairwise evolutionary distances from the aligned sequences. To obtain the confidence values for the 16S-rDNA-based genetic affiliations, the original sequence dataset was resampled 1000 times using SEQBOOT and subjected to bootstrap analysis. Phylogenetic trees were constructed using parsimony analysis (DNAPARS) and clustering algorithms such as FITCH, KITCH and the UPGMA linkage algorithm (Felsenstein, 1993). The different algorithms gave consistent results.
On the basis of 16S rDNA sequence similarity values, strain CMS 76orT was most closely related to members of the genus Kocuria (96·1–99·8 % similarity) and exhibited highest similarity values with the sequence of the type strain of K. rosea (99·8 %). In the 16S-rDNA-based phylogenetic tree (Fig. 1), strain CMS 76orT was observed to be positioned closest to K. rosea DSM 20447T. The statistical significance of this lineage was high (bootstrap value, 100 %). It was observed that the overall genomic relatedness between strains CMS 76orT and K. rosea ATCC 186T, based on DNA–DNA hybridization performed using the membrane filter method (Reddy et al., 2000b), was 71 %.
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), the fatty acid composition (Sato & Murata, 1988), the types of menaquinone (Collins et al., 1977; Dunphy et al., 1971), the polar lipids and the cell-wall sugars were done according to standard methods, as described previously (Komagata & Suzuki, 1987). Furthermore, pigments were extracted from lyophilized bacterial cell pellets, separated by TLC, and the absorption spectra of the individual pigments were recorded using a Hitachi 2000 spectrophotometer (Jagannadham et al., 1991). The G+C content (mol%) of the DNA of strain CMS 76orT was determined as described previously (Reddy et al., 2000).
The affiliation of strain CMS 76orT with the genus Kocuria was further supported by its chemotaxonomic properties, since in strain CMS 76orT, as in other species of the genus Kocuria, the peptidoglycan present was L-Lys–Ala3, of the A3
peptidoglycan type. MK-7(H2) (48 %) and MK-8(H2) (43 %) were the major menaquinones of strain CMS 76orT; MK-6(H2) (5·6 %) and MK-9(H2) (3·4 %) were the minor menaquinones. The major fatty acid was anteiso-C15 : 0 (70·6 %). Phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), phosphatidylinositol and an unidentified phospholipid were the polar lipids of strain CMS 76orT. The DNA G+C content of strain CMS 76orT was 72·5 mol% (Tables 1 and 2
). Strain CMS 76orT also contained six carotenoid pigments (with RF values of 0·92, 0·78, 0·61, 0·53, 0·46 and 0·32, respectively). Pigment characteristics of strain CMS 76orT are available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
Strain CMS 76orT, like other species of the genus Kocuria, was an aerobic, non-halophilic, catalase-positive, indole-negative micro-organism that was sensitive to penicillin, chloramphenicol, ampicillin and erythromycin (Schleifer, 1986; Rainey et al., 1997; Stackebrandt et al., 1995; Kovács et al., 1999). Furthermore, the chemotaxonomic characteristics of the strain and the topology of the 16S-rDNA-based phylogenetic tree (Fig. 1) clearly indicated that strain CMS 76orT was a member of the genus Kocuria. At the 16S rDNA level, strain CMS 76orT was closely related to all species of the genus Kocuria (96·1–99·8 % similarity), and exhibited maximum similarity with the 16S rDNA sequence of K. rosea ATCC 186T. DNA–DNA hybridization data for strain CMS 76orT and K. rosea ATCC 186T indicated 71 % similarity between the two strains. Despite the similarity at the DNA level between the novel strain and K. rosea ATCC 186T, strain CMS 76orT differed from K. rosea ATCC 186T, and from K. kristinae (ATCC 186T), K. varians (ATCC 15306T), K. rhizophila (DSM 11926T) and K. palustris (DSM 11025T), with respect to the following five phenotypic features: its ability to grow at 5 °C; its inability to grow in the presence of 5 % NaCl; its production of acid from D-mannitol and D-xylose; its high DNA G+C content (72·5 mol%) (Table 1). In addition to the above five differences, strain CMS 76orT differed from K. rosea ATCC 186T and K. rosea ATCC 187 in that it formed extremely mucoid colonies on ABM plates, produced acid from D-glucose and D-mannitol, and could utilize adonitol, D-melibiose, D-cellobiose, meso-inositol, lactose, pyruvate, inulin, D-raffinose, L-arginine, L-aspartic acid, L-glutamic acid, L-leucine or L-phenylalanine as the sole carbon source. It was unable to utilize L-aspargine as the sole carbon source, but could ferment lactose and sucrose (Table 1). Furthermore, it was observed that in strain CMS 76orT six carotenoid pigments were present, whereas in K. rosea ATCC 186T only five pigments were present (supplementary data; http://ijs.sgmjournals.org). It was also observed in the present study that the type strain of K. rosea (ATCC 186T) had MK-7(H2) (33·4 %) and MK-8(H2) (61·0 %) as its major menaquinones, with MK-9(H2) (5·6 %) as a minor component. In contrast, strain CMS 76orT had MK-7(H2) (48 %) and MK-8(H2) (43 %) as its major menaquinones and, in addition, small amounts of MK-6(H2) (5·5 %) and MK-9(H2) (3·4 %) (Table 1). It was also observed that when K. rosea ATCC 186T and strain CMS 76orT were cultured in ABM they exhibited distinct quantitative differences in their fatty acid compositions. For instance, in strain CMS 76orT, iso-C14 : 0, iso-C16 : 0 and C18 : 0 were decreased relative to K. rosea ATCC 186T, whereas C14 : 0, iso-C17 : 0 and iso-C18 : 0 were increased and C18 : 1 was also present (Table 2). Strain CMS 76orT could also be differentiated from the other species of Kocuria on the basis of some of its phenotypic characteristics (Table 1). Thus, on the basis of the similarities of strain CMS 76orT with respect to the generic characteristics of the genus Kocuria, and its distinct differences with respect to all recognized species of Kocuria, it is proposed that strain CMS 76orT be assigned to a novel species of the genus Kocuria, as Kocuria polaris.
Description of Kocuria polaris sp. nov.
Kocuria polaris (po.la'ris. M.L. adj. polaris of, or pertaining to, a pole).
Cells are coccoid (1·0–1·5 µm in diameter), occurring in pairs, tetrads or clusters. Non-motile, Gram-positive and aerobic. Colonies on peptone/yeast extract medium are 0·1–2·0 mm in diameter and appear smooth, round, uniformly edged, translucent, mucoid and orange in colour. Psychrophilic. Grows between 5 and 30 °C, with optimum growth at 20 °C. Grows in ABM adjusted to pH 7–12 and tolerates up to 2·9 % NaCl. Catalase- and lipase-positive. Negative for L-lysine decarboxylase, L-arginine decarboxylase, 
-galactosidase, L-arginine dihydrolase and levan formation. Negative results in the indole test, the methyl red test and the Voges–Proskauer test. Utilizes L-rhamnose, D-cellobiose, D-fructose, D-galactose, D-glucose, glycerol, D-mannose, D-xylose, acetate, sorbitol, L-glutamine, L-glycine, L-serine and L-threonine as sole carbon sources, but can not utilize meso-erythritol, melezitose, D-ribose, D-sorbose, citrate, dextran, lactic acid, dulcitol, sucrose, succinic acid, thioglycollate, D-trehalose, L-cysteine, L-histidine, L-isoleucine, L-lysine, L-proline, L-methionine, L-tyrosine, L-tryptophan, L-valine or -hydroxybutyric acid as sole carbon sources. Does not produce gas from D-glucose, D-fructose, L-arabinose, D-xylose, L-rhamnose, D-galactose, D-mannose, D-mannitol, lactose or sucrose. Acid is produced in the presence of D-fructose, D-galactose, lactose and sucrose. Oxidizes D-glucose, D-maltose, D-ribose, D-trehalose and D-xylose, but not L-arabinose or D-galactose. Ferments D-fructose, but not L-arabinose, D-galactose, D-glucose, D-maltose, D-ribose, D-trehalose or D-xylose. Other characteristics of the species can be found in Table 1. Sensitive to ampicillin, cefoperazone, co-trimoxazole, amikacin, cefazoline, ciprofloxacin, penicillin, vancomycin, tetracycline, lomefloxacin, erythromycin, roxithromycin, tobramycin, cephotaxime, lincomycin, chloramphenicol, streptomycin, kanamycin, norfloxacin, amoxycillin and cefuroxime, but resistant to nitrofurantoin, nalidixic acid and colistin. Cell-wall peptidoglycan is L-Lys–Ala3 (type A3). D-Galactose, D-glucose and D-ribose are the major cell-wall sugars. Menaquinones and fatty acid composition of the species are shown in Tables 1 and 2, respectively. Polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. Cells contain six water-insoluble pigments that are soluble in methanol; pigment production is not dependent on any specific growth conditions or on the composition of the medium. DNA G+C content is 72·5 mol%. Isolated from a cyanobacterial mat sample from McMurdo Dry Valley, Antarctica (77°33'3''S, 160°38'E). The type strain is CMS 76orT (=MTCC 3702T =DSM 14382T).
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ACKNOWLEDGEMENTS |
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