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Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Correspondence
Yi-Chueh Lin
linyichueh{at}yahoo.co.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of IAM 14817T is AB177868.
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MAIN TEXT |
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) differ from each other in menaquinone type, cell-wall sugars, G+C content of their DNA and physiological features (Lin et al., 2004). The genus Plantibacter was proposed to accommodate a Gram-positive, non-motile rod, with cell-wall peptidoglycan containing 2,4-L-DAB, D-glutamic acid, D-alanine and glycine, with the menaquinones MK-10 and MK-11 and with a G+C content of about 70 mol% (Behrendt et al., 2002). The type species is Plantibacter flavus, which was isolated from the phyllosphere of grasses. Strain IAM 14817 was originally deposited in 1967 by V. I. Kudriavzev as Arthrobacter sp. NCIMB 9991. The strain was grown on peptone/yeast extract agar supplemented with brain heart infusion [PY-BHI, comprising 1 % peptone, 0.2 % yeast extract, 0.2 % NaCl, 0.2 % D-glucose, 0.2 % brain heart infusion (Difco Laboratories), 1.5 % agar (pH 7.0)] and nutrient agar medium [0.5 % peptone, 0.3 % meat extract, 0.3 % NaCl, 1.5 % agar (pH 7.0)]. It was cultured aerobically at 30 °C. P. flavus DSM 14012T was used as the reference strain.
Gram staining was performed as described by Gerhardt et al. (1994). Cell morphology was determined using cells grown on PY-BHI agar. Each sample used for scanning electron microscopy (model S-4500; Hitachi) was prepared by fixing cells with 1 % glutaraldehyde, followed by dehydration using a graded acetone series and a Hitachi model HCP-2 critical point drying apparatus. Motility was determined by the hanging drop method. Unless otherwise indicated, all tests were performed at 25 °C. Catalase activity was determined by bubble formation in 3 % H2O2 solution. Oxidase activity was determined by the oxidation of 1 % tetramethyl-p-phenylenediamine on filter paper. Acid production from carbohydrates, assimilation of carbohydrates, nitrate reduction and hydrolysis of aesculin and gelatin were studied using API 50 CH and API 20NE (bioMérieux).
Cell walls were prepared from 
500 mg (dry weight) bacterial cells as described by Schleifer & Kandler (1972). Amino acids in an acid hydrolysate of the cell walls were identified by two-dimensional ascending chromatography on cellulose TLC plates (Tokyo Kasei Co.) by the method of Harper & Davis (1979) and by HPLC as phenylthiocarbamoyl derivatives with a model LC-6AD HPLC apparatus (Shimadzu Co.) equipped with a Wakopak WS-PTC column (Wako Pure Chemical Industries, 1989). Analysis of enantiomeric diamino acid isomers was performed according to Sasaki et al. (1998). Cell-wall sugars were analysed as described by Mikami & Ishida (1983). Fatty acids were extracted from dried cells, purified and examined as described previously (Yokota et al., 1993). The glycolate test was performed by the method of Uchida & Aida (1977).
Isolation and purification of chromosomal DNA and determination of DNA G+C content were performed by the methods of Takagi et al. (1993). DNA–DNA relatedness values were determined as described by Ezaki et al. (1989). Extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to the methods of Lin et al. (2004). The 16S rRNA gene was aligned with published sequences retrieved from DDBJ using CLUSTAL W (Thompson et al., 1994) and edited using BIOEDIT (Hall, 1999). The phylogenetic tree was constructed on the basis of the neighbour-joining method (Saitou & Nei, 1987); distances were estimated by the method of Jukes & Cantor (1969) using MEGA version 2.1 (Kumar et al., 2001). The GenBank/EMBL/DDBJ accession numbers used in this analysis are given in Fig. 1. Arthrobacter citreus DSM 20133T was used as an outgroup.
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-galactosidase and 
-galactosidase. Hydrolysis of aesculin was positive. Acid was produced from adonitol, rhamnose, inositol, maltose, lactose and melezitose, but not from ribose, L-sorbose, sorbitol, melibiose, inulin or raffinose. The physiological and biochemical characteristics are summarized in Table 1.
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). Cellular fatty acids detected were anteiso-C15 : 0 (57.7 % of total fatty acids), anteiso-C17 : 0 (18.3 %), iso-C16 : 0 (13.4 %), iso-C15 : 0 (4.9 %) and C16 : 0 (3.1 %). The peptidoglycan of strain IAM 14817T contained D-glutamate, glycine, D-alanine and 2,4-L-DAB in a molar ratio of 1.0 : 1.0 : 0.8 : 1.9. The cell-wall sugars contained rhamnose, 6-deoxytalose and fucose. The acyl type of the muramic acid in the peptidoglycan was acetyl (Table 2).
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). The level of 16S rRNA gene sequence similarity between strain IAM 14817T and P. flavus DSM 14012T was 99.7 %. DNA–DNA hybridization levels between strain IAM 14817T and P. flavus DSM 14012T were 49–55 %. Hence, strain IAM 14817T can be differentiated phenotypically from P. flavus DSM 14012T (Table 2). Based on the phenotypic and genotypic data, strain IAM 14817T is proposed as the type strain of a novel species within the genus Plantibacter, for which the name Plantibacter auratus sp. nov. is proposed.
Description of Plantibacter auratus sp. nov.
Plantibacter auratus (au.ra'tus. L. masc. part. adj. auratus gold-coloured, yellow).
Cells are Gram-positive, aerobic rods, 0.3–0.4x0.5–1.1 µm, non-sporulating and non-motile. Colonies are circular, convex, smooth and pale yellow on PY-BHI agar. Optimal temperature for growth is generally 30 °C. Catalase-positive, oxidase-negative. Positive for -galactosidase, -galactosidase and hydrolysis of aesculin. Acid is produced from adonitol, rhamnose, inositol, maltose, lactose and melezitose. The following tests are negative: reduction of nitrate or nitrite, nitrate respiration, alkaline phosphatase and production of acid from ribose, L-sorbose, sorbitol, melibiose, inulin and raffinose. The diamino acid in the cell wall is 2,4-L-DAB and the muramic acid in the peptidoglycan is of an acetyl type. The major fatty acid is 12-methyl tetradecanoic acid (anteiso-C15 : 0), followed by 14-methyl hexadecanoic acid (anteiso-C17 : 0), 14-methyl pentadecanoic acid (iso-C16 : 0) and hexadecanoic acid (C16 : 0). The predominant menaquinones are MK-9 and MK-10. The DNA G+C content is 68 mol%.
The type strain is IAM 14817T (=NCIMB 9991T=NBRC 15702T). The type strain was originally deposited in the NCIMB in 1967 by V. I. Kudriavzev as Arthrobacter sp. The source of the strain is unknown.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
Hall, T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Harper, J. J. & Davis, G. H. G. (1979). Two-dimensional thin-layer chromatography for amino acid analysis of bacterial cell walls. Int J Syst Bacteriol 29, 56–58.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kumar, S., Tamura, K., Jakobsen, I.-B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.
Lin, Y.-C., Uemori, K., de Briel, D. A., Arunpairojana, V. & Yokota, A. (2004). Zimmermannella helvola gen. nov., sp. nov., Zimmermannella alba sp. nov., Zimmermannella bifida sp. nov., Zimmermannella faecalis sp. nov. and Leucobacter albus sp. nov., novel members of the family Microbacteriaceae. Int J Syst Evol Microbiol 54, 1669–1676.
Mikami, H. & Ishida, Y. (1983). Post-column fluorometric detection of reducing sugars in high-performance liquid chromatography using arginine. Bunseki Kagaku 32, E207–E210.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasaki, J., Chijimatsu, M. & Suzuki, K. (1998). Taxonomic significance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov. Int J Syst Bacteriol 48, 403–410.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Takagi, H., Shida, O., Kadowaki, K., Komagata, K. & Udaka, S. (1993). Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov. Int J Syst Bacteriol 43, 221–231.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Uchida, K. & Aida, A. (1977). Acyl type of bacterial cell wall: its simple identification by colorimetric method. J Gen Appl Microbiol 23, 249–260.[CrossRef]
Wako Pure Chemical Industries (1989). Technical note on the system of PTC-amino acid analysis. Osaka: Wako Pure Chemical Industries, Ltd (in Japanese).
Yokota, A., Takeuchi, M., Sakane, T. & Weiss, N. (1993). Proposal of six new species in the genus Aureobacterium and transfer of Flavobacterium esteraromaticum Omelianski to the genus Aureobacterium as Aureobacterium esteraromaticum comb. nov. Int J Syst Bacteriol 43, 555–564.
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