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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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with glycolyl residues and a DNA G+C content of 66·5 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain US15T was closely related to Microbacterium arborescens IFO 3750T, Microbacterium imperiale IFO 12610T and Microbacterium ulmi LMG 20991T (96·9, 96·8 and 96·2 % similarities, respectively), and formed a separate lineage within the genus Microbacterium. Combined genotypic and phenotypic data showed that strain US15T (=DSM 16915T=KCTC 19080T) merits recognition as the type strain of a novel species within the genus Microbacterium, for which the name Microbacterium paludicola sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain US15T is AJ853909.
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MAIN TEXT |
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; Wong et al., 1988; Uffen, 1997). Current knowledge of microbial action on xylan has already led to suggestions about new technologies that are ready for development in both agriculture and industry (Kuhad & Singh, 1993).
During the course of our studies to screen micro-organisms that were able to degrade xylan from various environmental sources, a yellow-pigmented xylanolytic bacterium, strain US15T, was isolated from swamp forest soil and subjected to polyphasic taxonomic investigation. The results from phenotypic and genotypic characterizations suggested that the strain belongs to the genus Microbacterium, which was first described by Orla-Jensen (1919); its description was emended by Collins et al. (1983) and more recently it has been emended by Takeuchi & Hatano (1998) to unite the genera Microbacterium and Aureobacterium. At the time of writing, the genus Microbacterium consisted of 39 species with validly published names (type species is Microbacterium lacticum) that were isolated from various environmental sources including soil, plants, insects, air, dairy products, sewage, steep liquor and human clinical specimens.
A soil sample from swamp forest in Ulsan was collected and diluted serially in 0·85 % NaCl solution. Aliquots of each serial dilution were spread onto R2A agar (Difco) supplemented with insoluble chromogenic xylan (25 g l–1; Ten et al., 2004) and incubated at 28 °C for 7 days. A colony surrounded by a conspicuous clearing zone, designated US15T, was isolated and subcultivated onto nutrient agar (Difco) at 28 °C for 48 h for further analyses.
The Gram reaction was determined as described by Gerhardt et al. (1994). Cell morphology and motility were observed under a phase-contrast microscope (Optiphot; Nikon) at x1000 magnification, with cells grown for 1–7 days on nutrient agar. Oxidase activity was tested using 1 % tetramethyl-p-phenylenediamine (Tarrand & Groschel, 1982) and catalase activity was tested using 3 % H2O2. Growth was investigated on R2A agar, trypticase soy agar (TSA; BBL) and nutrient agar at temperatures ranging from 5 to 45 °C, at pH ranging from 4 to 9, in different salt concentrations (1, 2, 4 and 6·5 %) and on MacConkey agar (Difco). Hydrolysis of casein and starch was tested on casein agar and starch agar (Difco). A H2S production test was performed on triple-sugar-iron agar (BBL). Carbon-source utilization tests, acid-production tests and additional physiological tests were performed using API 20NE, API 32GN, API 50CH and API ZYM galleries according to the manufacturer's instructions (bioMérieux).
For the analysis of fatty acids, strain US15T was cultivated on TSA at 28 °C for 4 days. Microbacterium arborescens DSM 20754T (=IFO 3750T), Microbacterium imperiale DSM 20530T (=IFO 12610T) and Microbacterium ulmi LMG 20991T were used as reference strains under the same conditions.
Fatty acid methyl esters were prepared and analysed as described previously (Klatte et al., 1994) using the standard Microbial Identification system (MIDI) for automated gas chromatography analysis (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Isoprenoid quinones were extracted and purified as described previously (Tindall, 1990), and dried preparations were dissolved in 200 µl 2-propanol; 1–10 µl samples were separated by HPLC without further purification. Purified cell-wall preparations were obtained as described by Schleifer & Kandler (1972). Amino acids and peptides in cell-wall hydrolysates were analysed by two-dimensional TLC on cellulose plates by using solvent systems described by Schleifer & Kandler (1972). Cell-wall sugars were analysed according to the procedures of Staneck & Roberts (1974). Murein acyl type was determined by the colorimetric method of Uchida et al. (1999). Polar lipids were extracted, examined by two-dimensional TLC and identified by using published procedures (Minnikin et al., 1977).
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Rainey et al. (1996). The 16S rRNA gene sequence was aligned with published sequences retrieved from EMBL by using CLUSTAL X (Thompson et al., 1997) and was edited using BIOEDIT (Hall, 1999). A 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 DNA G+C content was determined by HPLC after hydrolysis, as described by Tamaoka & Komagata (1984), and non-methylated 
DNA (Sigma) was used as a standard. The resultant neighbour-joining tree topology was evaluated by the bootstrap analysis (Felsenstein, 1985) based on 1000 resampled datasets.
Strain US15T formed visible colonies (1–2 mm in diameter) on nutrient agar at 28 °C within 48 h. Good growth occurred at temperatures ranging from 15 to 37 °C, but no growth was observed at 5 °C or at temperatures above 45 °C within 14 days. Growth was observed at pH 6–9. The colonies were lemon-yellow, translucent and circular with entire edges. Cells were Gram-positive, non-motile, non-spore-forming, short rods (0·8–1·0x1·0–2·5 µm in size). Strain US15T differed significantly from M. arborescens, M. imperiale and M. ulmi, its closest phylogenetic neighbours, in terms of acid production, carbon-source utilization and substrate hydrolysis profiles as well as colony colour. Physiological and biochemical characteristics are summarized in Table 1 and the species description.
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type, (L-homoserine)-D-glu
glyD-Orn, with glycolyl residues. The cell-wall sugars were glucose, galactose, mannose, rhamnose and fucose. The polar lipids were composed of diphosphatidylglycerol, phosphatidylglycerol and unknown polar lipids including glycolipid and phospholipid. Strain US15T showed a different peptidoglycan type from M. arborescens and M. imperiale, and a different menaquinone profile from M. ulmi. Detailed chemotaxonomic characteristics are summarized in Table 2.
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), which also supported the placement of strain US15T in a novel species.
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Description of Microbacterium paludicola sp. nov.
Microbacterium paludicola (pa.lu.di'co.la. L. n. palus -udis swamp; L. suff. -cola from L. masc. or fem. n. incola inhabitant; N.L. masc. or fem. n. paludicola inhabitant of a swamp).
Cells are non-motile, non-spore-forming, short rods (0·8–1·0x1·0–2·5 µm in size). Gram-positive, oxidase-positive and catalase-positive. Good growth occurs on R2A agar, TSA and nutrient agar at 15–37 °C, but no growth is observed at 5 °C, temperatures above 45 °C or on MacConkey agar. Optimal growth temperature range is 25–30 °C and optimal pH range is 6–8. Growth occurs in the presence of 1, 2, 4 and 6·5 % NaCl. Colonies are lemon-yellow, translucent and circular with entire edges. Indole and H2S are not produced. Nitrate is not reduced to nitrite. Xylan, starch and aesculin are hydrolysed, but gelatin, casein and cellulose are not hydrolysed. Arginine dihydrolase and urease are produced, but ornithine decarboxylase and lysine decarboxylase are not produced. Methyl-red and Voges–Proskauer tests are negative. Acid is produced from glycerol, L-arabinose, D-xylose, galactose, glucose, fructose, mannose, rhamnose, mannitol, methyl 
-D-mannoside, aesculin, salicin, cellobiose, maltose, sucrose, trehalose, melezitose, starch, glycogen and D-turanose, but not from erythritol, D-arabinose, ribose, L-xylose, adonitol, methyl -D-xylose, sorbose, dulcitol, inositol, sorbitol, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, lactose, melibiose, inulin, raffinose, xylitol, gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. The following compounds are utilized as sole carbon sources: mannose, gluconate, adipate, mannitol, D-glucose, salicin, L-arabinose, propionate, valerate, 2-ketogluconate, rhamnose, D-sucrose, maltose, DL-lactate, 5-ketogluconate and glycogen. The following carbon sources are not utilized: malate, phenyl acetate, D-melibiose, L-fucose, D-sorbitol, caprate, citrate, histidine, 3-hydroxybutyrate, 4-hydroxybenzoate, L-proline, N-acetylglucosamine, D-ribose, inositol, itaconate, suberate, malonate, acetate, L-alanine, glycogen, 3-hydroxybenzoate and L-serine. According to the results from the API ZYM test, 2-naphthyl butyrate, 2-naphthyl caprylate, 2-naphthyl myristate, L-leucyl 2-naphthylamide, L-valyl 2-naphthylamide, 2-naphthyl phosphate (pH 5·4), naphthol-AS-BI-phosphate, 2-naphthyl -D-glucopyranoside and 6-bromo-2-naphthyl -D-mannopyranoside are hydrolysed, but 2-naphthyl phosphate (pH 8·5), L-cystyl 2-naphthylamide, N-benzoyl-DL-arginine 2-naphthylamide, N-glutaryl-phenylalanine 2-naphthylamide, 6-bromo-2-naphthyl -D-galactopyranoside, 2-naphthyl -D-galactopyranoside, naphthol-AS-BI--D-glucuronide, 6-bromo-2-naphthyl -D-glucopyranoside, 1-naphthyl N-acetyl--D-glucosaminide and 2-naphthyl -L-fucopyranoside are not hydrolysed. The major menaquinones are MK-12 (49·2 % of total quinones) and MK-11 (44·2 %); a small amount of MK-13 (6·7 %) is also present. The predominant fatty acids are 15 : 0-anteiso (63·9 %), 17 : 0-anteiso (14·4 %) and 16 : 0-iso (11·7 %). The cell-wall peptidoglycan is of the B2 type, (L-homoserine)-D-gluglyD-Orn, with glycolyl residues. The cell-wall sugars are glucose, galactose, mannose, rhamnose and fucose. The polar lipids are composed of diphosphatidylglycerol, phosphatidylglycerol and unknown polar lipids including glycolipid and phospholipid. The G+C content of the DNA is 66·5 mol%.
The type strain, US15T (=DSM 16915T=KCTC 19080T), was isolated from swamp forest soil in Ulsan, Korea.
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ACKNOWLEDGEMENTS |
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