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1 The Key Laboratory for Microbial Resources of Ministry of Education, P. R. China, Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, P. R. China
2 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
3 Department of Chemistry, Chuxiong Normal College, Chuxiong, Yunnan, 675000, P. R. China
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr or
wjli{at}ynu.edu.cn
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), the predominant menaquinone MK-9(H2) and a polar lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown glycolipids. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The G+C content of the genomic DNA was 63·8 mol%. Strain YIM 70010T exhibited a 16S rRNA gene sequence similarity of 99·6 % and DNA–DNA relatedness value of 56 % with Citricoccus muralis DSM 14442T. The phenotypic characteristics and DNA–DNA relatedness data indicate that strain YIM 70010T can be distinguished from C. muralis (DSM 14442T). Therefore, on the basis of the polyphasic taxonomic data presented, a novel species of the genus Citricoccus, Citricoccus alkalitolerans sp. nov. (type strain, YIM 70010T=CCTCC AA 203008T=DSM 15665T=KCTC 19012T) is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70010T is AY376164.
An electron micrograph of a cell of strain YIM 70010T and a dendrogram showing the relationship of the strain with its nearest phylogenetic neighbours are available as supplementary material in IJSEM Online.
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with a single species, Citricoccus muralis. Its representative strain was isolated from a medieval wall painting and comprises Gram-positive, non-motile, non-spore-forming coccoid cells and the aerobic type of metabolism. In this report, we present the description of the second species of the genus, for which we propose the name Citricoccus alkalitolerans sp. nov.
Strain YIM 70010T was isolated from a desert soil sample collected in eastern Egypt by using medium recommended by Sato et al. (1983) for isolation of alkaliphilic and alkaline-resistant micro-organisms. Sodium carbonate was sterilized separately and then added to the medium. The pH of the medium was 10–10·5; NaHCO3/Na2CO3 buffer was used to adjust the pH. Strain YIM 70010T was cultivated aerobically at 28 °C for 1 week. Cells for biochemical and molecular systematic analyses were grown in shake flasks (at about 150 r.p.m.) of trypticase soy broth medium (pH 8·0–9·0) at 28 °C for 1 week. Stock cultures were maintained at 4 °C using modified Sato's slants and as glycerol suspensions (20 %, v/v) at –20 °C.
Strain YIM 70010T was grown on PYES medium (Altenburger et al., 2002b) for observation of the cell and colony morphology. The growth temperature was tested at 4, 10, 20, 28, 37, 40 and 45 °C on the same medium. Motility of cells and pH and NaCl tolerance of the strain were determined as described by Altenburger et al. (2002a, b). Metabolic properties were determined using API Coryne and API ID 32 E test kits (bioMérieux) according to the manufacturer's instructions. Other physiological and biochemical tests were performed as described previously (Shirling & Gottlieb, 1966).
Strain YIM 70010T was an aerobic, Gram-positive, non-motile, non-spore-forming coccus, of about 0·5–0·8 µm in diameter (Fig. A, available as supplementary material in IJSEM Online). Colonies of strain YIM 70010T on PYES medium were similar to those of C. muralis DSM 14442T. Growth was observed at initial pH values of between 5·5 and 12·0, with the optimum at pH 8·0–9·0. Other physiological and biochemical properties are given in Table 1 and in the species description.
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and Schleifer & Kandler (1972) with the modification that TLC on cellulose was applied instead of paper chromatography. The N terminus of the interpeptide bridge was determined by dinitrophenylation according to Schleifer (1985). The quantitative analysis of amino acids in the total hydrolysates was done by GC and GC-MS as described by MacKenzie (1987) and Groth et al. (1996).
Menaquinones were isolated using the methods of Minnikin et al. (1984) and separated by HPLC (Kroppenstedt et al., 1981; Kroppenstedt, 1982). Polar lipids were extracted, examined by two-dimensional TLC and identified using published procedures (Minnikin et al., 1984). Fatty acid analysis was performed using standard methods (Sasser, 1990) and results were compared with the database of fatty acids in the MIDI Sherlock Microbial Identification system (MIDI Inc.).
The peptidoglycan of strain YIM 70010T contained Ala, Gly, Glu and Lys in a molar ratio of 1·6 : 1·2 : 2·5 : 1·0. Labelling by using 1-fluoro-2,4-dinitrobenzene revealed that glutamic acid represents the amino terminus of the interpeptide bridge. From these results and the two-dimensional TLC peptide pattern of partial cell-wall hydrolysates (data not shown), it was concluded that the peptidoglycan type of strain YIM 70010T was Lys–Gly–Glu, variation A4 (Schleifer & Kandler, 1972) (type A11.42 or A11.56 according to the DSMZ, 2001). Other chemotaxonomic characteristics of the strain are presented in the species description.
Genomic DNA was isolated and purified by the method of Marmur (1961). The DNA G+C base content of strain YIM 70010T was measured as 63·8 mol% by using the thermal denaturation method (Marmur & Doty, 1962).
The 16S rRNA gene sequence of the strain was amplified by PCR using conserved primers close to the 3' and 5' ends of the gene as described previously (Cui et al., 2001). Multiple alignments with sequences of actinobacteria of the family Micrococcaceae and calculations of levels of sequence similarity were carried out using CLUSTAL_X (Thompson et al., 1997). A phylogenetic tree was reconstructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980, 1983). The topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
An almost-complete 16S rRNA gene sequence (1480 bp) for strain YIM 70010T was obtained and subjected to a comparative analysis. Strain YIM 70010T was phylogenetically most closely related to C. muralis DSM 14442T, with a 16S rRNA gene sequence similarity value of 99·6 %. The phylogenetic tree (Fig. B) is available as supplementary material in IJSEM Online.
DNA–DNA hybridization between strain YIM 70010T and C. muralis DSM 14442T was carried out by applying the optical renaturation method (De Ley et al., 1970; Huss et al., 1983; Jahnke, 1992) under optimal hybridization conditions. The determined DNA–DNA relatedness value of 56 % (experiment repeated twice) was significantly lower than 70 %, which is considered to be the threshold value for the delineation of genomic species (Wayne et al., 1987).
On the basis of phylogenetic, morphological and chemotaxonomic evidence and its physiological and biochemical distinctiveness (Table 1
), it is proposed that strain YIM 70010T be classified as Citricoccus alkalitolerans sp. nov.
Description of Citricoccus alkalitolerans sp. nov.
Citricoccus alkalitolerans (al.ka.li'to.le.rans. Arabic article al the; Arabic n. qaliy ashes of saltwort; Gr. adj. tolerans tolerating; N.L. part. adj. alkalitolerans referring to the ability of the organism to tolerate alkaline media).
Cells are aerobic, Gram-positive, non-motile, non-spore-forming cocci, of about 0·5–0·8 µm in diameter. Colonies on PYES medium are light yellow, circular, entire, somewhat convex, opaque and approximately 1·5 mm in diameter after 24 h at 28 °C. Growth occurs between 10 and 37 °C with an optimal growth temperature of 28 °C; no growth is observed at 4 or 40 °C. Optimum growth pH and NaCl concentration are 8·0–9·0 and 0–5 %, respectively. Catalase-positive and oxidase-negative. Urease- and tyrosinase-negative. Tweens 20 and 80, casein and starch are not decomposed. H2S production and indole production are negative. Nitrate is not reduced to nitrite. Activities for lipase and -glucosidase are positive. Negative for ornithine decarboxylase, arginine dihydrolase, lysine decarboxylase, 
-glucuronidase, - and -galactosidase, N-acetyl--glucosaminidase and -glucosidase. The following substrates are utilized as sole carbon sources for growth (with no acid production): glucose, galactose, sucrose, arabinose, mannose, mannitol, maltose, starch, xylose, ribose, cellobiose, salicin, sorbitol, lactose, dextrin, amygdalin, glycine and lysine. Fructose, N-acetyl--glucosamine, L-alanine, -alanine, L-histidine, L-ornithine, L-tryptophan and arginine are not utilized. The peptidoglycan is of the Lys–Gly–Glu type (variation A4). The predominant menaquinone is MK-9(H2) and the cellular polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown glycolipids. The cellular fatty acids are anteiso-C15 : 0 (74·58 %), iso-C15 : 0 (13·14 %), anteiso-C17 : 0 (3·94 %), iso-C16 : 0 (1·78 %), C15 : 0 (1·69 %), iso-C14 : 0 (1·14 %), iso-C13 : 0 (0·3 %), iso-C17 : 0 (0·85 %), anteiso-C13 : 0 (0·9 %), C13 : 0 (0·14 %), C14 : 0 (0·68 %), C13 : 0 (0·14 %) and C16 : 0 (0·86 %). The DNA G+C content is 63·8 mol%.
The type strain, YIM 70010T (=CCTCC AA 203008T=DSM 15665T=KCTC 19012T), was isolated from an alkaline soil sample collected from the eastern desert of Egypt.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Altenburger, P., Kämpfer, P., Schumann, P., Vybiral, D., Lubitz, W. & Busse, H.-J. (2002b). Georgenia muralis gen. nov., sp. nov., a novel actinobacterium isolated from a medieval wall painting. Int J Syst Evol Microbiol 52, 875–881.[Abstract]
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