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1 The Key Laboratory for Microbial Resources of Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, People's Republic of China
2 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People's Republic of China
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Wen-Jun Li
wjli{at}ynu.edu.cn or
lihxu{at}ynu.edu.cn
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ABSTRACT |
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, variation L-lys–D-Asp, supported the placement of strain YIM 70177T within the genus Isoptericola. Galactose was detected as cell wall sugar. Phospholipids identified were phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol, and the predominant menaquinones were MK-9(H4) and MK-9(H2). The major fatty acids were ai-C15 : 0, C16 : 0, ai-C17 : 0 and i-C15 : 0. The DNA G+C content was 72·8 mol%. The low level of DNA–DNA relatedness (27·4 %) to I. variabilis DSM 10177T in combination with differentiating chemotaxonomic and physiological data demonstrated that the isolate YIM 70177T should be classified as representing a novel species of the genus Isoptericola. The name Isoptericola halotolerans sp. nov. is proposed, with strain YIM 70177T (=DSM 16376T=KCTC 19046T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70177T is AY789835.
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was reclassified as Isoptericola variabilis by Stackebrandt et al. (2004) because of differences in the 16S rRNA gene sequence (96·6 % similarity), cell-wall sugar (glucose rather than fucose) and in the amino acid composition of the peptidoglycan (L-lys–asp versus L-Lys–Ser–Asp) in comparison with Cellulosimicrobium cellulans (Schumann et al., 2001). Isolate YIM 70177T from saline soil sampled in China is here proposed as the type strain of a novel species of the genus Isoptericola, Isoptericola halotolerans sp. nov.
Strain YIM 70177T was isolated by using the dilution plating method on a modified Horikoshi medium (Horikoshi & Grant, 1998). This medium contained (l–1) 10·0 g glucose, 5·0 g peptone, 5·0 g yeast extract, 1·0 g K2HPO4.3H2O, 0·2 g MgSO4.7H2O, 200 g NaCl, 10·0 g Na2CO3 and 15·0 g agar. NaCl and sodium carbonate were sterilized separately before addition to the medium. The pH of the medium was adjusted to 10·0–10·5 using NaHCO3/Na2CO3 buffer. Strain YIM 70177T was maintained on ISP 5 agar slants containing 10 % (w/v) NaCl at 4 °C and as 20 % (w/v) glycerol suspensions at –20 °C. Biomass for chemical and molecular studies was obtained by cultivation in shaken flasks (about 150 r.p.m.) using modified ISP 5 broth [10 % (w/v) NaCl, pH 7·0] at 28 °C for 1 week.
Gram staining was carried out by the standard Gram reaction and was confirmed by using the KOH lysis test method (Cerny, 1978). Morphology and motility were examined by light microscopy (model BH 2; Olympus) and electron microscopy (JEM-1010 electron microscope) using cells from exponentially growing cultures. Colony morphology was observed on marine agar (MA), ISP 5 medium containing 10 % NaCl and trypticase soy agar (TSA) containing 10 % NaCl after incubation at 28 °C for 3 days. Colony colour was determined by comparing the cultures with the most suitable colour chips from the ISCC-NBS colour charts (Kelly, 1964). Growth temperature was tested at 4, 10, 28, 37, 40, 45 and 55 °C on the same medium. pH and NaCl tolerances were examined as described by Tang et al. (2003). Metabolic properties were determined using API ID 32 E test kits (bioMérieux) according to the manufacturer's instructions. Other physiological and biochemical tests were performed as described by Gonzalez et al. (1978).
Growth of strain YIM 70177T was aerobic and cells stained Gram-positive. Coccoid and rod-shaped cells were observed, but neither flagella nor spores were found. Pale-yellow colonies with a smooth surface and a maximal diameter of approximately 1 mm were formed on MA and TSA containing 10 % NaCl after incubation for 48 h at 28 °C.
Strain YIM 70177T grew well at 28 and 37 °C; only slow growth occurred at 4 and 45 °C. Growth was observed at initial pH values between 6 and 10 and on TSA containing 0–25 % NaCl. The strain grew optimally at 28 °C, at pH 8–9 and in the presence of 10 % NaCl. Detailed physiological and biochemical characteristics of the strain are given in Table 1 and in the species description below.
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. The peptidoglycan type was A4, variation L-lys–D-Asp. Galactose was detected in the purified cell wall. Phospholipids identified were phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol. The menaquinones were MK-9(H4), MK-9(H2), MK-8(H4) and MK-9 (ratio of peak areas, 15 : 10 : 2 : 1). The major fatty acids were ai-C15 : 0, C16 : 0, ai-C17 : 0 and i-C15 : 0; the detailed profile is given in the species description.
Extraction of genomic DNA and PCR amplification of the 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 16S rRNA gene 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) using MEGA version 2.1 (Kumar et al., 2001). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
The 16S rRNA gene sequence (1382 bp) for strain YIM 70177T was determined. Phylogenetic analysis of strain YIM 70177T revealed that it was most closely related to I. variabilis DSM 10177T with a 16S rRNA gene sequence similarity of 98·3 %, and the two strains formed a distinct subclade in the family Promicromonosporaceae (Fig. 1).
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. DNA–DNA relatedness was studied using the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) by using a UV-VIS spectrophotometer model UV1601 (Shimadzu). The level of DNA–DNA relatedness between YIM 70177T and I. variabilis DSM 10177T was 27·4 % (repeated twice; the original recorded values were 25·8 and 29·0 %).
The results of 16S rRNA gene sequence analysis clearly demonstrate that strain YIM 70177T represents a member of the genus Isoptericola. Similarities in morphological characteristics, peptidoglycan type and DNA G+C content to I. variabilis DSM 10177T support the inclusion of strain YIM70177T within the genus Isoptericola. However, strain YIM 70177T differs from I. variabilis DSM 10177T in components of the cell-wall sugars, phospholipids, menaquinones, cellular fatty acids and some physiological and enzymic properties (see Table 1
). The level of DNA–DNA relatedness of 27·4 % between YIM 70177T and I. variabilis DSM 10177T is significantly lower than 70 %, the threshold value considered for the delineation of genomic species (Wayne et al., 1987). These results support the proposal of a novel species, for which the name Isoptericola halotolerans sp. nov. is proposed.
Description of Isoptericola halotolerans sp. nov.
Isoptericola halotolerans (ha.lo.to'le.rans. Gr. n. hals salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans salt-tolerating, referring to the organism's ability to tolerate high salt concentrations).
Cells are Gram-positive, coccoid or rod-shaped, non-motile and do not form spores. Primary mycelium is formed. Colonies are pale-yellow, circular, opaque and approximately 1·0 mm in diameter after 24 h growth at 28 °C. Optimal growth occurs at 10 % NaCl, pH 8·0–9·0 and 28 °C. In addition to the properties listed in Table 1, the strain is negative for gelatin liquefaction, ammonia production, milk peptonization and starch hydrolysis, but positive for methyl red test and nitrate reduction. The following substrates are utilized: glucose, ribose, arabinose, maltose, cellobiose, trehalose, sorbitol, lactose, xylose and dextrin. The peptidoglycan type is A4, variation L-lys–D-Asp. The cell-wall sugar is galactose. Phospholipids are phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinones are MK-9(H4) and MK-9(H2). The cellular fatty acids are ai-C15 : 0 (54·46 %), C16 : 0 (20·05 %), ai-C17 : 0 (10·69 %), i-C15 : 0 (7·02 %), C14 : 0 (2·33 %), i-C16 : 0 (1·44 %), C15 : 0 (0·98 %), C17 : 0 (0·61 %), C18 : 0 (0·36 %), i-C14 : 0 (0·64 %), i-C17 : 0 (0·51 %) and ai-C15 : 1 (0·51 %). The G+C content of the DNA of the type strain is 72·8 mol%.
The type strain, YIM 70177T (=DSM 16376T=KCTC 19046T), was isolated from a saline soil sample collected from Qinghai Province, north-west China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cerny, G. (1978). Studies on aminopeptidase for the distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5, 113–122.[CrossRef]
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