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1 Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, People's Republic of China
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 Botany Department, Faculty of Science, Cairo University, Beni-Suef, Egypt
4 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People's Republic of China
Correspondence
Wen-Jun Li
wjli{at}ynu.edu.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70003T is DQ059617.
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MAIN TEXT |
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by taxonomic dissection of the genus Micrococcus, and was clearly separated from Micrococcus and Micrococcus-related taxa on the basis of phylogenetic analyses using 16S rRNA gene sequences. Members of Kocuria are Gram-positive, aerobic, non-encapsulated, non-halophilic, non-endospore-forming cocci characterized by the presence of menaquinones MK-7(H2) and MK-8(H2), lysine-based peptidoglycan variation A3
, phosphatidylglycerol and diphosphatidylglycerol as the major phospholipids, a predominance of saturated branched fatty acids such as ai-C15 : 0 and a genomic DNA G+C content in the range 60–75 mol% (Stackebrandt et al., 1995; Bohá
ek et al., 1969; Kloos et al., 1974; Kocur et al., 1971; Kovács et al., 1999; Reddy et al., 2003; Kim et al., 2004). At the time of writing, there are eight Kocuria species with validly published names: Kocuria kristinae, K. palustris, K. polaris, K. rhizophila, K. rosea, K. varians, K. marina and K. carniphila (Stackebrandt et al., 1995; Kovács et al., 1999; Reddy et al., 2003; Kim et al., 2004; Tvrzová et al., 2005). Of these species, only K. marina was isolated from a high-salinity environment (Kim et al., 2004). In this paper, we report a polyphasic taxonomic study of strain YIM 70003T isolated from a saline, alkaline, desert-soil sample collected in Egypt.
Strain YIM 70003T was isolated on modified Horikoshi agar medium (Horikoshi & Grant, 1998) using the dilution plating method. This medium contained the following (g l–1): glucose, 10·0; peptone, 5·0; yeast extract, 5·0; KH2PO4, 1·0; MgSO4.7H2O, 0·2; Na2CO3, 10·0; and agar, 15; pH, 10·0–10·5. Sodium carbonate was sterilized separately and then added to the medium. NaHCO3/Na2CO3 buffer was used to adjust the pH. The purified strain was maintained on Horikoshi agar slants at 4 °C and as 20 % (w/v) glycerol suspensions at –20 °C. Biomass for chemical and molecular studies was obtained by cultivation using Horikoshi broth (28 °C, 1 week, 150 r.p.m.).
Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test (Cerny, 1978). Morphology and motility were examined by using light microscopy (model BH 2; Olympus) and electron microscopy (JEM-1010; JEOL) with cells from exponentially growing cultures. Colony morphology was observed on Horikoshi medium after incubation at 28 °C for 3 days. The colony colour was determined using ISCC–NBS colour charts (Kelly, 1964). Growth was tested in Horikoshi broth at 4, 10, 20, 28, 37, 40, 45 and 55 °C. The pH growth range and optimum were investigated at pH 4·0–13·0 using the buffer system described by Xu et al. (2005). Liquid cultures were cultivated in tubes at 28 °C for 2–3 weeks using ISP 2 as the basic medium. Growth at different concentrations (0, 1, 3, 7, 10, 15 and 20 %) of sodium, potassium, magnesium and calcium chlorides was tested, again in ISP 2 basic medium. Metabolic properties were determined using API ID 32E test kits (bioMérieux) according to the manufacturer's instructions. Other physiological and biochemical tests were performed as described previously (Gonzalez et al., 1978).
The cells of strain YIM 70003T were Gram-positive, aerobic, non-motile, non-endospore-forming, coccoid and about 0·8–1·1 µm in diameter. The colonies were pink, circular, slightly convex, opaque and had a maximum diameter of about 2 mm after incubation at 28 °C for 48 h on Horikoshi agar medium. No diffusible pigments were produced on any of the media. The isolate was catalase-positive and gave a negative oxidase reaction. Detailed physiological and biochemical properties are given in Table 1 and in the species description.
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). Strain YIM 70003T possessed peptidoglycan type Lys–Ala3, variation A3. Galactose and minor amounts of glucose were detected in the purified cell wall. The phospholipids consisted of phosphatidylglycerol and diphosphatidylglycerol. The menaquinones were represented by MK-8(H2), MK-9(H2), MK-7(H2), MK-6(H2) and MK-10(H2) (27 : 8·5 : 2·5 : 1·5 : 1, respectively). The major fatty acids were ai-C15 : 0 (55·3 %) and i-C15 : 0 (20·4 %).
Extraction of genomic DNA and PCR amplification of 16S rRNA gene were performed as described by Xu et al. (2003). Multiple alignments with sequences of the most closely related actinobacteria and calculations of levels of sequence similarity were carried out using CLUSTAL X (Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980, 1983) by using MEGA version 2.1 (Kumar et al., 2001). The topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
The almost-complete 16S rRNA gene sequence (1492 bp) for strain YIM 70003T was determined. Phylogenetic analysis revealed that the strain's closest relative were K. polaris DSM 14382T and K. rosea DSM 20447T, showing respective 16S rRNA gene sequence similarities of 98·6 and 98·2 % (Fig. 1). The DNA G+C content was determined as 73·0 mol% by using the thermal denaturation method of Marmur & Doty (1962). DNA–DNA relatedness was studied using the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) on a UV-Vis spectrophotometer (model UV1601; Shimadzu). The DNA–DNA hybridization values for strain YIM 70003T with respect to K. polaris DSM 14382T and K. rosea DSM 20447T were 56·6 and 15·5 %, respectively.
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The DNA–DNA relatedness among strains YIM 70003T, K. polaris DSM 14382T and K. rosea DSM 20447T is below 70 %, which indicates that the novel isolate represents a distinct genospecies (Wayne et al., 1987) of the genus Kocuria. The relatively large proportion of menaquinone MK-9(H2) (21 %), the fatty acid profile and some other phenotypic properties (e.g. pigmentation, NaCl and temperature tolerance, oxidase reaction, nitrate reduction and hydrolysis results) of strain YIM 70003T differentiate it from other members of the genus Kocuria at the phenotypic level (Table 1
).
On the basis of its phenotypic and genotypic properties, it is proposed that strain YIM 70003T represents a novel species of the genus Kocuria, for which the name Kocuria aegyptia sp. nov. is proposed.
Description of Kocuria aegyptia sp. nov.
Kocuria aegyptia (ae.gyp'ti.a. L. fem. adj. aegyptia from Egypt, referring to the country of isolation of the type strain).
Cells are Gram-positive, coccoid, occur in pairs, tetrads or clusters, are non-motile and do not form endospores. Colonies are pink, circular, opaque and approximately 2 mm in diameter. Cannot grow in ISP 2 medium without salt, but can grow in ISP 2 medium containing 1–5 % NaCl, 1–10 % KCl, 1–5 % MgCl2.6H2O or 1–5 % CaCl2; optimum growth occurs at 3 %, but at 5 % in the case of KCl. The temperature range for growth is 20–40 °C, with optimum growth occurring at 28 °C. The pH range for growth is 5·0–12·0, with optimum growth occurring between at pH 10·0–10·5. The oxidase test with tetramethyl-p-phenylenediamine is negative. Negative for urease, N-acetylglucosaminidase, L-aspartic arylamidase, 
-galactosidase, -galactosidase, -maltosidase, -glucuronidase, Tweens 20 and 80, esterase, tyrosinase, in methyl red and Voges–Proskauer tests, for melanin, indole and H2S production, nitrate reduction, gelatin liquefaction, milk peptonization and coagulation and for starch hydrolysis; the catalase reaction is positive. Produces ornithine decarboxylase, arginine dihydrolase, lysine decarboxylase, lipase, -glucosidase and ammonia. Maltose, D-glucose, D-cellobiose, D-trehalose, D-sorbitol, D-fructose, D-mannose and dextrin can each be utilized as a sole carbon source; acid is produced only from D-fructose. The peptidoglycan type is Lys–Ala3, variation A3. The cell-wall sugars consist mainly of galactose and minor amounts of glucose. The phospholipids are phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinones are MK-8(H2) and MK-9(H2). The major cellular fatty acids are ai-C15 : 0 (55·3 %) and i-C15 : 0 (20·4 %). The G+C content of the DNA is 73·0 mol%.
The type strain is YIM 70003T (=CCTCC AA203006T=CIP 107966T=KCTC 19010T=DSM 17006T) and was isolated from a saline, alkaline, desert-soil sample collected from Egypt.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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