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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
2 Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
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9c (18.8 %). Strain LW6T has the major respiratory menaquinones MK-8(H4) and MK-8(H2) and polar lipids phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and unknown glycolipid/phospholipids. The cell wall peptidoglycan of strain LW6T contained the amino acids ornithine, lysine, glutamic acid, alanine, glycine and aspartic acid. Its molar DNA G+C content is 69 mol% (Tm). Analysis of 16S rRNA gene sequences indicated that strain LW6T was related phylogenetically to members of the genus Ornithinimicrobium, with similarities ranging from 98.3 to 98.7 %. The DNA–DNA relatedness of strain LW6T to Ornithinimicrobium humiphilum DSM 12362T and Ornithinimicrobium kibberense K22-20T was respectively 31.5 and 15.2 %. Based on these results, it is concluded that strain LW6T represents a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium pekingense sp. nov. is proposed. The type strain is strain LW6T (=CGMCC 1.5362T =JCM 14001T).
A scanning electron micrograph of cells of strain LW6T, two-dimensional thin-layer chromatograms of the polar lipids of strain LW6T and cellular fatty acid profiles of strain LW6T and related type strains are available as supplementary material with the online version of this paper.
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) was proposed on the basis of 16S rRNA gene sequence analysis within the suborder Micrococcineae and its description was later emended (Stackebrandt & Schumann, 2000). Chemotaxonomic studies have indicated that genera within the family Intrasporangiaceae have different characteristic amino acid residues of their cell-wall peptidoglycan; for example, Intrasporangium has LL-diaminopimelic acid, Janibacter has meso-diaminopimelic acid and Ornithinicoccus and Ornithinimicrobium have ornithine. The genus Ornithinimicrobium (Groth et al. 2001) was first established for description of a soil actinomycete with L-ornithine in the peptidoglycan, Ornithinimicrobium humiphilum. A second species, Ornithinimicrobium kibberense, was also isolated from soil and has been described recently (Mayilraj et al., 2006). Although these two Ornithinimicrobium species were from soil samples, Ornithinimicrobium-like strains have been detected widely, in marine sediments [Ornithinimicrobium sp. CNJ824 (GenBank accession no. DQ448703) and Ornithinimicrobium sp. JL1084 (DQ985059)], in an aquatic system (nine strains; DQ923280–DQ923288) and in batch-fed garbage compost (Ornithinimicrobium sp. TUT1205; AB188211; Narihiro et al., 2004). In this note, we report the isolation of strain LW6T from a bioreactor for wastewater treatment and its identification as a novel member of the genus Ornithinimicrobium.
Bacterial strain LW6T was isolated from activated sludge of a sequential batch reactor treating wastewater containing various nitroaromatic compounds (nitrobenzene, nitrophenol, 2,4-dinitrophenol) and aniline. The reactor had been operated for a year at the time when the sludge was sampled, and the performance of the reactor has been reported elsewhere (Liu et al., 2007). The sludge sample was suspended in sterile water by using vigorous vortexing and a portion of the suspension was spread directly on LB agar plates. The plates were incubated at 30 °C for about 1 week. Single colonies on the plates were picked up and strain LW6T was obtained by repeatedly streaking the culture on new plates from a single colony.
Routine cultivation was conducted at 30 °C with TSA and TSB media. Gram reactions were determined according to the method described by Gerhardt et al. (1994). Cell flagellation and morphology were examined by transmission electron microscopy and scanning electron microscopy. The growth temperature range was determined with a TN3F temperature-gradient incubator (Advantec). Catalase and oxidase activities, Voges–Proskauer reaction, aerobic production of acids from carbohydrates and further biochemical characterization were performed according to the methods of Barrow & Feltham (1993) and Wieser et al. (2002). Utilization of carbon sources (10 g l–1) was tested in nutrient broth with the following ingredients (g l–1): (NH4)2SO4, 2; NaH2PO4, 0.5; MgSO4 . 7H2O, 0.2; CaCl2 . 2H2O, 0.1; KH2PO4, 0.5. For determination of growth, culture was incubated at 37 °C for 4 days and then the OD600 was measured (OD600>2, growth; 1<OD600<2, weak growth; OD600<1, no growth).
Cells of strain LW6T were Gram-positive, irregular short rods and cocci with a size range of 0.5–0.8x1.0–1.6 µm (see Supplementary Fig. S1 available in IJSEM Online). Catalase was produced but oxidase was not. Colonies were light yellow, smooth, circular and 0.2–1.0 mm in diameter after 3 days incubation. Growth was observed at a temperature range of 26–38 °C and a pH range of 6–9, with optimal growth at 33–37 °C and pH 7.8–8.2. Anaerobic growth of strain LW6T was not observed. Growth at an NaCl concentration of 7 % (w/v) was observed. Strain LW6T used a range of sugars as carbon sources for growth, but production of acids from sugars was rarely detected. More physiological and biochemical characteristics of strain LW6T are provided in the species description and in Table 1. Strain LW6T is clearly different from Ornithinimicrobium humiphilum and Ornithinimicrobium kibberense in growth at pH 9.0 and at 42 °C, tolerance of 7 % NaCl and hydrolysis of Tween 80. Strain LW6T differs further phenotypically from Ornithinimicrobium humiphilum and Ornithinimicrobium kibberense in assimilation of various carbon sources (Table 1).
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). Cellular fatty acids were extracted, methylated and analysed by using the Sherlock Microbial Identification System according to the manufacturer's instructions. Menaquinones were extracted and purified by the method described by Collins (1985) and were analysed by HPLC (Wu et al., 1989) with various menaquinones and ubiquinones (Hu et al., 2001) as references. Polar lipids were analysed as described and cited by Feng et al. (2005). The results showed that the cell-wall peptidoglycan of strain LW6T contained the amino acids ornithine, lysine, glutamic acid, alanine, glycine and aspartic acid. The most abundant cellular fatty acids of strain LW6T were iso-methyl-branched acids (iso-C15 : 0, 38.9 %; iso-C17 : 19c, 18.8 %) and the anteiso-methyl-branched acid anteiso-C15 : 0 (6.6 %). A complete list of all fatty acids detected and a comparison with those of Ornithinimicrobium humiphilum HKI 0124T and Ornithinimicrobium kibberense K22-20T is provided in Supplementary Table S1. Strain LW6T has a similar cellular fatty acid profile to Ornithinimicrobium humiphilum (Groth et al., 2001) and Ornithinimicrobium kibberense (Mayilraj et al., 2006), but it differs quantitatively. Strain LW6T had MK-8(H4) (76.8 %) and MK-8(H2) (13.7 %) as major respiratory quinones; MK-8 and MK-6 were also detected in small amounts (each less than 10 %). This menaquinone pattern is different from that of Ornithinimicrobium humiphilum [in which MH-8(H2), MK-8 and MK-6 were detected in minor amounts] and Ornithinimicrobium kibberense [only MK-8(H4) was reported]. The polar lipids consisted of phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and unknown glycolipid/phospholipids (Supplementary Fig. S2). The DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962); the G+C content of strain LW6T was determined to be 69 mol%.
The nearly complete 16S rRNA gene of strain LW6T (1396 bp) was amplified and sequenced according to a method described by Zhang et al. (2003). Alignment of the 16S rRNA gene sequence with those sequences exhibiting high identities and deposited in the public databases was performed using the CLUSTAL_X program, version 1.64b (Thompson et al., 1997). Based on 16S rRNA gene sequence identities, strain LW6T is closely related to Ornithinimicrobium humiphilum DSM 12362T (98.7 %), Ornithinimicrobium kibberense K22-20T (98.3 %), Serinicoccus marinus JC1078T (94.5 %) and Ornithinicoccus hortensis HKI 0125T (94.5 %). A phylogenetic tree (Fig. 1) was constructed based on evolutionary distances calculated with the Kimura two-parameter model. Alignment positions with insertions or deletions were excluded from the calculations. The tree in Fig. 1 indicated that strain LW6T clustered with species belonging to the genus Ornithinimicrobium, and this cluster was strongly supported (99 %).
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. Results showed that the DNA–DNA relatedness of strain LW6T to Ornithinimicrobium humiphilum DSM 12362T and Ornithinimicrobium kibberense K22-20T was 31.5 and 15.2 %, respectively. Therefore, it is concluded that strain LW6T represents a novel species of the genus Ornithinimicrobium, and the name Ornithinimicrobium pekingense sp. nov. is proposed.
Description of Ornithinimicrobium pekingense sp. nov.
Ornithinimicrobium pekingense (pe.king.en'se. N.L. neut. adj. pekingense pertaining to Peking, the former English name of Beijing, where the type strain was isolated and studied).
Cells are mainly irregular short rods and cocci with a size range of 1.0–1.6x0.5–0.8 µm and are non-motile. Gram-positive, aerobic and heterotrophic. Colonies are light yellow, smooth and circular with entire margins. Growth is observed over a temperature range of 26–38 °C and pH range of 6–9. Optimal growth occurs at 33–37 °C and the optimal pH is 7.8–8.2. Catalase and lipase are produced, but oxidase is not. Nitrate is reduced and starch, citrate, benzoate and gelatin are hydrolysed. Growth occurs at 7 % NaCl (w/v). The Voges–Proskauer reaction and casein hydrolysis are negative. Acid production from the following carbohydrates is negative: melezitose, D-mannose, melibiose, cellobiose, glucose, D-ribose, sucrose, D-fructose, D-xylose, D-galactose, maltose, rhamnose, sorbitol, raffinose, D-lactose and L-arabinose. Alkali production from the following carbohydrates is positive: melibiose, salicin, rhamnose, D-mannose and D-lactose. Utilization of various substrates as sole carbon sources is detailed in Table 1
. The predominant menaquinone is MK-8(H4), and a significant amount of MK-8(H2) is present. The major cellular fatty acids are iso-methyl-branched acids (iso-C15 : 0, 38.9 %; iso-C17 : 19c, 18.8 %). Polar lipids consist of phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and unknown glycolipid/phospholipids. The G+C content of the DNA is 69 mol% (Tm).
The type strain is LW6T (=CGMCC 1.5362T =JCM 14001T), isolated from activated sludge of a bioreactor treating wastewater containing various chlorinated nitroaromatic compounds.
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ACKNOWLEDGEMENTS |
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Collins, M. D. (1985). Isoprenoid quinone analysis in classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
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Zhang, D., Yang, H., Zhang, W., Huang, Z. & Liu, S.-J. (2003). Rhodocista pekingensis sp. nov., a cyst-forming phototrophic bacterium from a municipal wastewater treatment plant. Int J Syst Evol Microbiol 53, 1111–1114.
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