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1 The Key Laboratory for Microbial Resources of Ministry of Education, People's Republic of China, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, People's Republic of China
2 Department of Chemistry, Chuxiong Normal College, Chuxiong, Yunnan 675000, People's Republic of China
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Cheng-Lin Jiang
lihxu{at}ynu.edu.cn or
liact{at}hotmail.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains YIM 70084T and YIM 70097T are AY226508 and AY226510.
Images of cells of strains YIM 70084T and YIM 70097T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) and in the reclassification of Micrococcus halobius Onishi and Kamekura 1972 as Nesterenkonia halobia (Stackebrandt et al., 1995). Recently, a second species of the genus Nesterenkonia, Nesterenkonia lacusekhoensis, has been proposed by Collins et al. (2002). In this work, we present the polyphasic taxonomic characterization of two halotolerant strains of the genus Nesterenkonia that were isolated from hypersaline soil samples from Xinjiang Province, western China.
Strains YIM 70084T and YIM 70097T were isolated using a modified glycerol/asparagine agar (ISP 5) medium (Shirling & Gottlieb, 1966) supplemented with 15 % (w/v) MgCl2.6H2O and KCl, respectively. The isolation plates were incubated at 28 °C for 2 weeks. The purified strains were cultivated and maintained on medium containing 0·1 % (w/v) asparagine, 1 % glycerol, 0·1 % K2HPO4.3H2O, 0·5 % yeast extract, 10 % MgCl2.6H2O (for YIM 70084T) or 10 % KCl (for YIM 70097T). The pH was adjusted to 7·2 with 1 M NaOH. When required, the medium was solidified with 2 % (w/v) agar. Biomass for chemical and molecular systematic studies was grown in shaken flasks (
150 r.p.m.) at 28 °C for 1 week. Morphological properties were examined by light microscopy (Olympus microscope BH-2) and transmission electron microscopy with a Hitachi model H-800 TEM. Media and procedures used for determination of physiological features and carbon source utilization were those described by Shirling & Gottlieb (1966). The colony colour of strains grown on medium PYGV (Staley, 1968) and modified ISP 5 agar medium was determined by comparing the cultures with the most suitable colour chips from the ISCC-NBS colour charts (Kelly, 1964).
Motility of cells was studied on LB swarming agar (0·3 %, w/v). The methods for measuring pH, temperature and salt tolerance were described by Tang et al. (2003). Some metabolic properties of the strains were determined by using API Coryne system with API ID 32 E test kits (bioMérieux) according to the manufacturer's instructions.
The sugars of purified cell walls were analysed as described by Stanek & Roberts (1974). Purified peptidoglycan preparations were obtained using the method described by Schleifer & Kandler (1972). Amino acids and peptides in cell-wall hydrolysates were analysed by two-dimensional ascending TLC on cellulose plates (Merck) using the solvent systems of Schleifer & Kandler (1972). The amino-terminal amino acid of the interpeptide bridge was determined by dinitrophenylation as described by Schleifer (1985). Molar ratios of amino acids were determined by GC and GC-MS of N-heptafluorobutyryl amino acid isobutyl esters (MacKenzie, 1987). Analysis of enantiomers of peptidoglycan amino acids was performed by GC of N-pentafluoropropionyl amino acid isopropyl esters (Frank et al., 1980) on an L-chirasil Val column (Macherey-Nagel) as described by Groth et al. (1997). Phospholipid analysis was carried out as described by Komagata & Suzuki (1987). Menaquinones were isolated using the method of Collins et al. (1977) and were analysed by HPLC (Groth et al., 1997). Cellular fatty acid composition was performed as described by Sasser (1990) using the Microbial Identification System (MIDI Inc.).
DNA for base composition was prepared following the method of Marmur (1961) and the G+C content was determined using the thermal denaturation method of Marmur & Doty (1962) by using a UV-VIS spectrophotometer model UV1601 (Shimadzu). Extraction of genomic DNA and amplification of 16S rDNA were performed as described by Xu et al. (2003). Multiple alignments with sequences of a broad selection of Actinobacteria 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 the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
Young cells (24–48 h) of YIM 70084T were Gram-positive, non-spore-forming and motile cocci with flagella. Agar colonies were light orange–yellow to deep orange–yellow and their surface was smooth. The strain grew optimally in modified ISP 5 medium at 28 °C, at pH 7·0–8·0 and in the presence of 10·0 % (w/v) MgCl2.6H2O. Cells of strain YIM 70097T were Gram-positive, non-motile, non-spore-forming, irregular rods. The colony colour was light yellow and the surface of colonies was smooth. It grew optimally in modified ISP 5 medium at 28 °C, at pH 8·0–9·0 and in the presence of 10·0 % KCl. Detailed physiological and biochemical characteristics of the two strains are given in Table 1 and in the species descriptions.
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(Schleifer & Kandler, 1972), with L-lysine in a tetrapeptide subunit and an interpeptide bridge consisting of Gly–Asp. Its cell-wall sugars were xylose and galactose and the menaquinone composition was MK-7, MK-8, MK-5, MK-6, MK-9 (ratio 80 : 11 : 5 : 1 : 0·5). Its polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The cellular fatty acids were ai-C15 : 0 (51·33 %), i-C16 : 0 (17·32 %), ai-C17 : 0 (13·74 %), ai-C15 : 1 (9·97 %), C14 : 0 (0·33 %), C16 : 0 (3·26 %), i-C14 : 0 (1·25 %), i-C15 : 0 (1·12 %), ai-C13 : 0 (0·35 %), i-C15 : 1 (0·37 %), i-C16 : 1 (0·49 %) and ai-C17 : 1
9c (0·49 %). Strain YIM 70097T differed from strain YIM 70084T in displaying the interpeptide bridge Gly–L-Glu. Its cell-wall sugars were ribose and galactose and the menaquinone composition was MK-8, MK-7 and MK-9 (ratio 56 : 24 : 17). The phospholipid pattern contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. The cellular fatty acids contained ai-C15 : 0 (28·50 %), ai-C17 : 0 (38·10 %), C15 : 0 (0·26 %), C16 : 0 (1·26 %), i-C14 : 0 (0·31 %), i-C15 : 0 (6·50 %), i-C16 : 0 (8·97 %), i-C17 : 0 (4·06 %), i-C19 : 0 (0·26 %), i-C15 : 1 (0·98 %), i-C16 : 1 (3·48 %), ai-C15 : 1 (0·43 %) and ai-C17 : 19c (6·9 %). The DNA base compositions of strains YIM 70084T and YIM 70097T were 64·4 and 66·7 mol% G+C.
The lengths of the almost-complete 16S rDNA sequences analysed for strains YIM 70084T and YIM 70097T were 1490 and 1506 bp. The two isolates showed 96·9 % 16S rDNA sequence similarity to each other and represented a separate cluster within the genus Nesterenkonia. Whereas strain YIM 70097T showed 96·8 % sequence similarity to both type strains of validly published Nesterenkonia species, the sequence similarities of strain YIM 70084T to N. halobia DSM 20541T and N. lacusekhoensis DSM 12544T were respectively 96·5 and 96·3 %. A phylogenetic tree is shown in Fig. 1.
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). It is therefore evident from the phylogenetic data and the great difference in phenotypic characteristics (Table 1
) that the two isolates from hypersaline soils represent two previously unknown species. Thus, the novel species Nesterenkonia halotolerans sp. nov. (type strain YIM 70084T) and Nesterenkonia xinjiangensis sp. nov. (type strain YIM 70097T) are proposed.
Description of Nesterenkonia halotolerans sp. nov.
Nesterenkonia halotolerans (ha.lo.to'le.rans. Gr. n. halos salt; L. part. tolerans tolerating; N.L. pres. part. halotolerans referring to the ability to tolerate high salt concentrations).
Cells are Gram-positive, non-spore-forming, motile cocci (see Supplementary Figure in IJSEM Online). The colony colour on modified ISP 5 medium is deep orange–yellow (the colour of the fringe) to light orange–yellow (the colour of the centre); some colonies resemble concentric rings. Colonies are circular, opaque and approximately 2·5–3·5 mm in diameter after 24 h at 28 °C. The optimum growth temperature is 28 °C. The optimum concentration of MgCl2.6H2O is 10 %. The type strain is positive for gelatin liquefaction and urease production and negative for milk peptonization and coagulation, nitrate reduction, growth on cellulose, H2S and melanin production and starch hydrolysis. The following substrates are utilized: glucose, galactose, mannose, fructose, sucrose, maltose, starch, lactose and dextrin. Ribose, arabinose, cellobiose, trehalose, sorbitol and xylose are not utilized. The peptidoglycan type is A4, L-Lys–Gly–Asp. Cell-wall sugars are xylose and galactose. Main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. Predominant menaquinones are MK-7 and MK-8. Major cellular fatty acids are ai-C15 : 0, i-C16 : 0 and ai-C17 : 0. The DNA G+C content of the type strain is 64·4 mol%.
The type strain is YIM 70084T (=CCTCC AA001022T=DSM 15474T), isolated from a saline soil sample from Xinjiang Province, China.
Description of Nesterenkonia xinjiangensis sp. nov.
Nesterenkonia xinjiangensis (xin.ji.ang.en'sis. N.L. fem. adj. xinjiangensis pertaining to Xinjiang, the province of western China in which the samples were collected).
Cells are Gram-positive, non-motile, non-spore-forming, diphtheroid, irregular rods (see Supplementary Figure in IJSEM Online). The colony colour on modified ISP 5 medium is light yellow. Colonies are circular, opaque, somewhat convex and approximately 3·5–4·5 mm in diameter after 24 h at 28 °C. The optimum growth temperature is 28 °C. The optimum concentration of KCl for growth is 10·0 %. The type strain is positive for gelatin liquefaction, milk peptonization and urease production and negative for milk coagulation, nitrate reduction, growth in cellulose, H2S and melanin production and starch hydrolysis. Almost all tested carbon sources, including glucose, galactose, mannose, fructose, sucrose, maltose, starch, lactose, dextrin, ribose, arabinose, cellobiose and xylose, are utilized; trehalose and sorbitol are not utilized. The peptidoglycan type is A4, L-lys–gly–L-Glu. The cell-wall sugars are ribose and galactose. Predominant menaquinones are MK-8, MK-7 and MK-9. Main phospholipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. Major cellular fatty acids are ai-C15 : 0 and ai-C17 : 0. The DNA G+C content of the type strain is 66·7 mol%.
The type strain is YIM 70097T (=CCTCC AA001025T=DSM 15475T), isolated from a saline soil sample from Xinjiang Province, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D., Lawson, P. A., Labrenz, M., Tindall, B. J., Weiss, N. & Hirsch, P. (2002). Nesterenkonia lacusekhoensis sp. nov., isolated from hypersaline Ekho Lake, East Antarctica, and emended description of the genus Nesterenkonia. Int J Syst Evol Microbiol 52, 1145–1150.[Abstract]
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