SGM Journals
Actinobacteria

Kocuria aegyptia sp. nov., a novel actinobacterium isolated from a saline, alkaline desert soil in Egypt

Wen-Jun Li, Yu-Qin Zhang, Peter Schumann, Hua-Hong Chen, Wael N. Hozzein, Xin-Peng Tian, Li-Hua Xu, Cheng-Lin Jiang

  • 1Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, People's Republic of China
  • 2DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
  • 3Botany Department, Faculty of Science, Cairo University, Beni-Suef, Egypt
  • 4Institute 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

    • International Journal of Systematic and Evolutionary Microbiology 2006; 56(4):733–737 · https://doi.org/10.1099/ijs.0.63876-0

    View at publisher PubMed

    Abstract

    A coccoid, non-motile actinobacterium, designated strain YIM 70003T, was isolated from a saline, alkaline, desert-soil sample from Egypt. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism formed a distinct phyletic line within the genus Kocuria and was most closely related to Kocuria polaris DSM 14382T (98·6 % sequence similarity) and Kocuria rosea DSM 20447T (98·2 %). Chemotaxonomic data, including the Lys–Ala3 peptidoglycan type, the presence of phosphatidylglycerol and diphosphatidylglycerol as the predominant phospholipids, the presence of MK-8(H2) and MK-9(H2) as the major menaquinones, the predominance of fatty acids ai-C15 : 0 and i-C15 : 0 and the DNA G+C content, also supported the affiliation of the isolate to the genus Kocuria. The low DNA–DNA relatedness with K. polaris DSM 14382T (56·6 %) and K. rosea DSM 20447T (15·5 %) in combination with phenotypic data show that strain YIM 70003T should be classified as a novel species of the genus Kocuria. The name Kocuria aegyptia sp. nov. is proposed, with strain YIM 70003T (=CCTCC AA203006T=CIP 107966T=KCTC 19010T=DSM 17006T) as the type strain.

    • Published online ahead of print on 2 December 2005 as DOI 10.1099/ijs.0.63876-0.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70003T is DQ059617.

    The genus Kocuria was established by Stackebrandt et al. (1995) 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 A3α, 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; Boháček 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.

    Table 1.

    Differentiating characteristics of K. aegyptia YIM 70003T and other Kocuria species

    Species/strains: 1, K. rosea; 2, K. varians; 3, K. kristinae (data in columns 1–3 taken from Stackebrandt et al., 1995); 4, K. palustris (Kovács et al., 1999); 5, K. rhizophila (Kovács et al., 1999); 6, K. polaris (Reddy et al., 2003 unless indicated); 7, K. marina (Kim et al., 2004); 8, K. carniphila (Tvrzová et al., 2005); 9, K. aegyptia sp. nov. YIM 70003T (this study). +, Positive; −, negative; v, variable; w, weak; nd, no data available. Data for all species except K. rosea, K. varians and K. kristinae are based on the type strains.

    The chemotaxonomic properties, including peptidoglycan type, cell-wall sugars, phospholipids, menaquinones and whole-cell fatty acid pattern, were analysed as described previously (Li et al., 2004). Strain YIM 70003T possessed peptidoglycan type Lys–Ala3, variation A3α. 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.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic dendrogram obtained by distance matrix analysis of 16S rRNA gene sequences, showing the position of strain YIM 70003T among its phylogenetic neighbours.Numbers on branch nodes are bootstrap percentages (1000 resamplings); only values over 50 % are shown. The sequence of Streptomyces megasporus DSM 41476T (Z68100) was used as a root (not shown). Bar, 1 % sequence divergence.

    The results of comparative 16S rRNA sequence analysis clearly demonstrate that strain YIM 70003T is a member of the genus Kocuria. The chemotaxonomic characteristics of strain YIM 70003T, such as peptidoglycan type, major fatty acids and DNA G+C content, were consistent with its assignment to the genus Kocuria.

    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, β-galactosidase, α-galactosidase, α-maltosidase, β-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, β-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 A3α. 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.

    Acknowledgments

    The authors are grateful to Professor Reiner M. Kroppenstedt and Dr R. Pukall for kindly providing type strains. This research was supported by the National Basic Research Program of China (project no. 2004CB719601), the National Natural Science Foundation of China (project no. 30270004) and the Yunnan Provincial Natural Science Foundation (project no. 2004 C0002Q). W.-J. L. was also supported by the Program for New Century Excellent Talent in University (NCET).

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