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1 State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Zhong-Guan-Cun, Haidian, Beijing 100080, China
2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
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ABSTRACT |
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A scanning electron micrograph of cells of strain LW4T and a table detailing its cellular fatty acid composition are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, several emendations to this description have been made based on utilization of glucose, G+C content of the genomic DNA and phylogenetic analysis of the 16S rRNA gene (detailed in Wieser et al., 2002). Although the genus comprises only three recognized species at the time of writing, Micrococcus luteus, Micrococcus lylae (Stackebrandt et al., 1995; Kloos et al., 1974) and Micrococcus antarcticus (Liu et al., 2000), the isolation and detection of Micrococcus-like bacteria from activated sludge have been reported repeatedly (Painter, 1983; Kataoka et al., 1996; Wieser et al., 2002). In this study, we report the isolation from activated sludge and the identification of a novel Micrococcus-like isolate, strain LW4T.
Bacterial strain LW4T was isolated from activated sludge of a sequential batch reactor treating mixed wastewater of various nitroaromatic compounds (nitrobenzene, nitrophenol, 2,4-dinitrophenol) and aniline. The reactor had been operated for 1 year at the time when the sludge was sampled, and the performance of the reactor was highly efficient (removal rates 
99 %) in removing all nitroaromatic compounds and aniline. 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 bacterial strain LW4T was obtained by repeatedly streaking the culture on new plates from a single colony.
Routine cultivation was conducted at 30 °C with LB media. Gram reactions were determined according to the method described by Gerhardt et al. (1994). Cell motility and morphology were examined by transmission electron microscopy and scanning electron microscopy (see Supplementary Fig. S1 in IJSEM Online). Growth temperature range was determined with a TN3F temperature-gradient incubator (Advantec). Catalase and oxidase activities, the Voges–Proskauer reaction, aerobic production of acids from carbohydrates, carbon source utilization and other biochemical characterization were performed according to the methods of Barrow & Feltham (1993) and Wieser et al. (2002).
Cells of strain LW4T were Gram-positive cocci, with a diameter range of 0.7–1.0 µm. Flagella were not observed. Colonies were yellow, smooth, circular and 0.5–1.5 mm in diameter after 3 days cultivation on LB agar. Strain LW4T was aerobic and grew over the temperature range 26–34 °C and pH range 5–9. Optimal growth was observed at 30.5–31.5 °C and pH 6.0–6.2. Strain LW4T was not able to use nitrobenzene, nitrophenol, 2,4-dinitrophenol or aniline for growth. Additional physiological and biochemical properties of strain LW4T are provided in the species description below.
Biomass for chemotaxonomic analyses was cultivated according to Stackebrandt et al. (1995) and at 30 °C for 24 h. Cell-wall analysis was performed according to the method described by Hasegawa et al. (1983). Cellular fatty acids were extracted, methylated and analysed by using the Sherlock Microbial Identification System following the manufacturer's instructions. Menaquinones were extracted and purified according to the method of Collins (1985) and were analysed by HPLC (Wu et al., 1989), with a previously characterized mixture of various menaquinones and ubiquinones (Hu et al., 2001) as a reference. The results indicated that the cell-wall peptidoglycan of strain LW4T contained lysine, glutamic acid, alanine, glycine and aspartic acid. The most abundant cellular fatty acids of strain LW4T were anteiso-C15 : 0 (32.15 %) and iso-C15 : 0 (31.65 %), which were also found to be the dominant cellular fatty acids of other members of the genus Micrococcus (Wieser et al., 2002). However, a significant amount of iso-C15 : 1 was detected in strain LW4T (19.21 %), compared with only trace amounts in other Micrococcus species (Wieser et al., 2002). Detailed information on the cellular fatty acid composition of strain LW4T is provided in Supplementary Table S1. Strain LW4T had MK-8(H2) (57.3 %) as the major respiratory quinone, plus a significant of amount MK-7(H2) (32.9 %).
DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962), with Escherichia coli K-12 as reference. The DNA G+C content of strain LW4T was 71.4 mol%.
The nearly complete 16S rRNA gene of strain LW4T (1403 bp) was amplified and sequenced as described by Zhang et al. (2003). Alignments of 16S rRNA gene sequences were performed with the CLUSTAL_X program, version 1.64b (Thompson et al., 1997). A neighbour-joining phylogenetic tree (Fig. 1) was constructed based on evolutionary distances that were calculated with the Kimura two-parameter model. Alignment positions with insertions or deletions were excluded from the calculations. 16S rRNA gene sequence analysis indicated that strain LW4T was phylogenetically related to members of the genus Micrococcus, with similarities ranging from 96.5 to 97.3 %. The phylogenetic tree (Fig. 1) also indicated that strain LW4T clustered with Micrococcus species and that this cluster was strongly supported (100 %).
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), such as the ability to reduce nitrate, assimilation of various carbon resources and major respiratory quinones. To distinguish strain LW4T from other Micrococcus species further, levels of DNA–DNA relatedness of strain LW4T to the type species of recognized Micrococcus species were determined by using the method described by Huß et al. (1983). Levels of DNA–DNA relatedness of strain LW4T to M. luteus DSM 20030T, M. lylae DSM 20315T and M. antarcticus AS 1.2372T were 55, 48 and 36 %, respectively.
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Description of Micrococcus flavus sp. nov.
Micrococcus flavus (fla'vus. L. masc. adj. flavus yellow, pertaining to the yellow colour of the colonies).
Cells are spherical, 0.7–1.0 µm in diameter and non-motile. Gram-positive, aerobic and heterotrophic. Colonies are yellow, smooth and circular with entire margins. Optimal growth occurs at 30.5–31.5 °C and pH 6–6.2. Catalase- and oxidase-positive. Starch is hydrolysed. Negative for the Voges–Proskauer reaction, lipase, reduction of nitrate and utilization of citric acid. Gelatin is not hydrolysed. No acid production from carbohydrates. Glycerol, trehalose and dextrin are used as carbon sources, but D-arabinose, fructose, mannitose, rhamnose, melibiose, xylitol, malic acid, L-glutamic acid, L-lactic acid, nitrilosides, L-arabinose, cellobiose, D-lactose, D-glucose, inositol, maltose, D-mannose, D-melibiose, raffinose, D-ribose, salicin and sorbitol are not. The predominant menaquinones are MK-8(H2) and MK-7(H2). The major cellular fatty acids are anteiso-C15 : 0 (32.15 %) and iso-C15 : 0 (31.65 %). The G+C content of the DNA is 71.4 mol% (Tm).
The type strain, LW4T (=CGMCC 1.5361T=JCM 14000T), was isolated from activated sludge in a bioreactor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cohn, F. (1872). Untersuchungen über Bakterien. Beitr Biol Pflanz 1, 127–244 (in German).
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.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). Methods for General and Molecular Bacteriology. Washington DC: American Society for Microbiology.
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
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Kataoka, N., Tokiwa, T., Tanaka, Y., Takeda, K. & Suzuki, T. (1996). Enrichment culture and isolation of slow growing bacteria. Appl Microbiol Biotechnol 45, 771–777.[CrossRef]
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Liu, H., Xu, Y., Ma, Y. & Zhou, P. (2000). Characterization of Micrococcus antarcticus sp. nov., a psychrophilic bacterium from Antarctica. Int J Syst Evol Microbiol 50, 715–719.[Abstract]
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Painter, H. A. (1983). Metabolism and physiology of aerobic bacteria and fungi. In Ecological Aspects of Used-Water Treatment: Biological Activities and Treatment Processes, vol. 2, pp. 11–75. Edited by C. R. Curds & H. A. Hawks. London: Academic Press.
Stackebrandt, E., Koch, C., Gvozdiak, O. & Schumann, P. (1995). Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 emend. Int J Syst Bacteriol 45, 682–692.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Wieser, M., Denner, E. B. M., Kämpfer, P., Schumann, P., Tindall, B., Steiner, U., Vybiral, D., Lubitz, W., Maszenan, A. M. & other authors (2002). Emended descriptions of the genus Micrococcus, Micrococcus luteus (Cohn 1872) and Micrococcus lylae (Kloos et al. 1974). Int J Syst Evol Microbiol 52, 629–637.[Abstract]
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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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