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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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). Subsequently, two further Cellulosimicrobium species, Cellulosimicrobium variabile (Bakalidou et al., 2002) and Cellulosimicrobium funkei (Brown et al., 2006), were described. However, Cellulosimicrobium variabile was reclassified within a novel genus, Isoptericola, as Isoptericola variabilis (Stackebrandt et al., 2004). In this study, we report on the taxonomic characterization of a bacterial strain, DS-61T, which is phylogenetically closely related to the genus Cellulosimicrobium.
Strain DS-61T was isolated from a soil sample collected from Dokdo (3 ° 14' 12'' N 13 ° 52' 07'' E), Korea, by means of the standard dilution plating technique performed at 25 °C on 10x diluted nutrient agar (Difco). Cellulosimicrobium cellulans DSM 43879T and Cellulosimicrobium funkei DSM 16025T, which were used as reference strains, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). The morphological, physiological and biochemical characteristics of strain DS-61T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 25 °C. The cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–40 °C) was measured on TSA. Growth in the absence of NaCl and at various NaCl concentrations (0.5 and 1.0–10.0 %, w/v, using increments of 1.0 %) was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was included. The pH range for growth was determined in nutrient broth (Difco) that had been adjusted, prior to sterilization, to various pH values (pH 4.5–10.5, using increments of 0.5 pH units) by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and on TSA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and the hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). The hydrolysis of aesculin and the reduction of nitrate were determined as described by Lanyi (1987). The utilization of substrates as sole carbon and energy sources was tested according to the method of Kämpfer et al. (1991). Susceptibility to antibiotics was tested on TSA plates using discs containing the following antibiotics: polymyxin B (100 U), streptomycin (50 µg), penicillin G (20 U), chloramphenicol (100 µg), ampicillin (10 µg), cephalothin (30 µg), gentamicin (30 µg), novobiocin (5 µg), tetracycline (30 µg), kanamycin (30 µg), lincomycin (15 µg), oleandomycin (15 µg), neomycin (30 µg) and carbenicillin (100 µg). Other physiological properties and enzyme activities were tested by using the API 20E and API ZYM systems (bioMérieux).
Cell biomass for DNA extraction and for analysis of the cell-wall components, isoprenoid quinones and polar lipids was obtained from cultures grown, with shaking at 150 r.p.m., in trypticase soy broth (Difco) at 25 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). The sequencing of the amplified 16S rRNA gene and the phylogenetic analysis were performed as described by Yoon et al. (2003). The DNA G+C content was determined according to the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed and the resultant nucleotides analysed by reversed-phase HPLC. The presence or absence of diaminopimelic acid in the peptidoglycan was determined according to the method described by Komagata & Suzuki (1987). Preparation of the cell walls and determination of the peptidoglycan structure were carried out by using the methods of Schleifer & Kandler (1972) and MacKenzie (1987) with the modification that TLC on cellulose was applied instead of paper chromatography. Whole-cell sugars were determined as described by Komagata & Suzuki (1987). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and were identified by two-dimensional TLC followed by spraying with the appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). For fatty acid methyl ester analysis, cell mass of strain DS-61T was harvested from TSA plates after incubation for 7 days at 25 °C, and cell mass of Cellulosimicrobium cellulans DSM 43879T and Cellulosimicrobium funkei DSM 16025T was harvested from TSA plates after incubation for 3 days at 28 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). DNA–DNA hybridization was performed fluorometrically according to the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed using five replications for each sample: the highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as the DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain DS-61T are given in the species description (see below) or are shown in Table 1. The almost-complete 16S rRNA gene sequence of strain DS-61T determined in this study comprised 1478 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree based on the neighbour-joining algorithm, strain DS-61T joined the cluster comprising Cellulosimicrobium cellulans and Cellulosimicrobium funkei with a bootstrap confidence value of 94.9 % (Fig. 1). The relationship between strain DS-61T and these two Cellulosimicrobium species was also recovered in trees based on the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DS-61T exhibited 16S rRNA gene sequence similarity values of 97.4 and 97.6 % with respect to the type strains of Cellulosimicrobium cellulans and Cellulosimicrobium funkei, respectively.
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, showed that strain DS-61T contained lysine, alanine, threonine, aspartic acid and glutamic acid in the approximate ratio 0.5 : 3.4 : 0.7 : 1.0 : 1.0. The reduced amounts of lysine and threonine are due to the occurrence of the stable peptide L-Lys–L-Thr. Under more extreme conditions (120 °C, 6 M HCl, 16 h), the peptidoglycan of strain DS-61T contained lysine, alanine, threonine, aspartic acid and glutamic acid in the approximate ratio 0.9 : 2.4 : 1.0 : 1.0 : 1.0. From these data, it was concluded that strain DS-61T contains peptidoglycan of the A4
type, based on L-Lys–L-Thr–D-Asp, as described by Schleifer & Kandler (1972). Galactose was the only whole-cell sugar detected in strain DS-61T. The predominant isoprenoid quinone detected in strain DS-61T was a tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)]. The fatty acid profile of strain DS-61T comprised large amounts of straight-chain and branched fatty acids; the major components (>10 % of total fatty acids) were anteiso-C15 : 0 and iso-C15 : 0 (Table 2). The major polar lipids detected in strain DS-61T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. These fatty acid and polar lipid profiles were similar to those of Cellulosimicrobium species (Schumann et al., 2001; Stackebrandt et al., 2004; Brown et al., 2006). The DNA G+C content of strain DS-61T was 72.9 mol%.
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). There are no distinct phenotypic, particularly chemotaxonomic, properties that serve to differentiate strain DS-61T from the genus Cellulosimicrobium (Schumann et al., 2001; Brown et al., 2006). Accordingly, it is appropriate to place strain DS-61T within the genus Cellulosimicrobium on the basis of phylogenetic and chemotaxonomic relatedness (Schumann et al., 2001; Brown et al., 2006). Strain DS-61T exhibited mean DNA–DNA relatedness values of 19 and 15 % with respect to Cellulosimicrobium cellulans DSM 43879T and Cellulosimicrobium funkei DSM 16025T, respectively. Strain DS-61T differs from these two recognized Cellulosimicrobium species in some phenotypic respects, as shown in Table 1. The phylogenetic and genetic distinctiveness and differential phenotypic properties of DS-61T are sufficient to categorize it as a member of a species that is distinct from these two recognized Cellulosimicrobium species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, strain DS-61T represents a novel species of the genus Cellulosimicrobium, for which the name Cellulosimicrobium terreum sp. nov. is proposed.
Emended description of the genus Cellulosimicrobium Schumann et al. 2001
The description of the genus Cellulosimicrobium is as given by Schumann et al. (2001) and Brown et al. (2006), but with the following amendments. The cell-wall peptidoglycan type is A4, based on L-Lys–D-Ser–D-Asp or L-Lys–L-Thr–D-Asp. The DNA G+C contents are in the range 72.9–74.5 mol%.
Description of Cellulosimicrobium terreum sp. nov.
Cellulosimicrobium terreum (ter're.um. L. neut. adj. terreum of the earth).
Cells are Gram-positive, non-spore-forming rods or cocci (0.4–0.8x0.4–2.0 µm); in older cultures, cells are Gram-variable and most cells are cocci. Colonies on TSA are circular, convex, smooth, glistening, yellow in colour and 1.5–2.0 mm in diameter after 7 days incubation at 25 °C. Substrate hyphae are present. Optimal temperature for growth is 25 °C. Growth occurs at 4 and 34 °C, but not at 35 °C. Optimal pH for growth is 6.5–7.5; growth occurs at pH 6.0 and 9.0, but not at pH 5.5 or 9.5. Growth occurs in the presence of 0–9 % (w/v) NaCl; optimal growth occurs in the presence of 1.0 % (w/v) NaCl. Anaerobic growth does not occur on TSA or on TSA supplemented with nitrate. Oxidase-negative. Tweens 20, 40, 60 and 80 are hydrolysed. H2S and indole are not produced. L-Glutamate is utilized as a sole carbon and energy source, but succinate, benzoate and formate are not. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Susceptible to cephalothin, chloramphenicol, neomycin, novobiocin, oleandomycin, penicillin G, streptomycin and tetracycline, but not to carbenicillin, gentamicin, kanamycin, lincomycin or polymyxin B. The cell-wall peptidoglycan type is L-Lys–L-Thr–D-Asp. The only whole-cell sugar is galactose. The predominant menaquinone is MK-9(H4). Major fatty acids are anteiso-C15 : 0 and iso-C15 : 0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The DNA G+C content of the type strain is 72.9 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DS-61T (=KCTC 19206T=DSM 18665T), was isolated from soil from Dokdo, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, J. M., Steigerwalt, A. G., Morey, R. E., Daneshvar, M. I., Romero, L.-J. & McNeil, M. M. (2006). Characterization of clinical isolates previously identified as Oerskovia turbata: proposal of Cellulosimicrobium funkei sp. nov. and emended description of the genus Cellulosimicrobium. Int J Syst Evol Microbiol 56, 801–804.
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
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.
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MacKenzie, S. L. (1987). Gas chromatographic analysis of amino acids as the N-heptafluorobutyryl isobutyl esters. J Assoc Off Anal Chem 70, 151–160.[Medline]
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
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Schumann, P., Weiss, N. & Stackebrandt, E. (2001). Reclassification of Cellulomonas cellulans (Stackebrandt and Keddie 1986) as Cellulosimicrobium cellulans gen. nov., comb. nov. Int J Syst Evol Microbiol 51, 1007–1010.[Abstract]
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Stackebrandt, E., Schumann, P. & Cui, X.-L. (2004). Reclassification of Cellulosimicrobium variabile Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov. Int J Syst Evol Microbiol 54, 685–688.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rRNA gene sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
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-glucuronidase, 