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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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with glycolyl residues and a DNA G+C content of 69·7 mol%. Phylogenetic analysis, based on 16S rRNA gene sequencing, showed that strain S3-ET is most similar to Microbacterium hominis IFO 15708T and Microbacterium foliorum DSM 12966T (97·6 and 97·4 % sequence similarity, respectively), and that it forms a separate lineage with M. hominis in the genus Microbacterium. DNA–DNA hybridization results and phenotypic properties showed that strain S3-ET could be distinguished from all known Microbacterium species and represented a novel species, for which the name Microbacterium xylanilyticum sp. nov. is proposed; the type strain is S3-ET (=DSM 16914T=KCTC 19079T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Microbacterium xylanilyticum S3-ET is AJ853908.
A phase-contrast micrograph of S3-ET cells and an extended version of the 16S rRNA gene phylogenetic tree are available as supplementary figures in IJSEM Online.
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MAIN TEXT |
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). Xylan-degrading enzymes, such as -1,4-D-xylan xylanohydrolase and -1,4-D-xylan xylohydrolase, are produced mainly by a wide range of micro-organisms and are used in the food and pulp industries (Dekker & Richards, 1976; Wong et al., 1988; Christov & Prior, 1993; Coughlan & Hazlewood, 1993).
During the screening of xylan-degrading bacteria from various sources, we isolated a strain, S3-ET, from the biofilm of a membrane bioreactor for wastewater treatment. Polyphasic taxonomic studies showed that this strain belonged to the genus Microbacterium. The genus Microbacterium was first described by Orla-Jensen (1919), was emended by Collins et al. (1983) and was more recently emended, to unite the genera Microbacterium and Aureobacterium, by Takeuchi & Hatano (1998). At the time of writing, the genus Microbacterium accommodates 39 species with validly published names (type species, Microbacterium lacticum), which have been isolated from various environmental sources including soil, plants, air, dairy products, sewage, steep liquor and human clinical specimens.
A biofilm sample from a membrane bioreactor was collected and diluted serially in 0·85 % saline solution. Aliquots of each serial dilution were spread on 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 with a conspicuous halo was isolated and subcultivated on nutrient agar (Difco) at 28 °C for 48 h for further analyses; it was designated as strain S3-ET.
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 1000x 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 (BBL) and nutrient agar, at temperatures ranging from 5 to 45 °C, at pH values 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). An 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 50 CH and API ZYM galleries according to the instructions of the manufacturer (bioMérieux).
For the analysis of fatty acids, strain S3-ET was cultivated on trypticase soy agar at 28 °C for 48 h. Microbacterium hominis DSM 12509T (=IFO 15708T) and Microbacterium foliorum DSM 12966T 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 chromatographic analysis (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Isoprenoid quinones were extracted and purified as previously described (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 using the solvent systems described by Schleifer & Kandler (1972). Cell-wall sugars were analysed according to the procedures of Staneck & Roberts (1974). The murein acyl type was determined by using 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), which were 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 using the method of Jukes & Cantor (1969) with MEGA, version 2.1 (Kumar et al., 2001). The DNA G+C content was determined by using HPLC after hydrolysis, as described by Tamaoka & Komagata (1984), and non-methylated 
DNA (Sigma) was used as a standard. DNA–DNA hybridization to determine genomic relatedness was performed fluorometrically by the method of Ezaki et al. (1989), using photobiotin (A1935; Sigma)-labelled DNA probes and 96-well microdilution wells (Greiner Bio-One).
Strain S3-ET formed visible colonies (about 1 mm in diameter) on nutrient agar at 28 °C within 48 h. Good growth occurred at temperatures ranging from 15 to 37 °C, but growth was weak at 5 °C and no growth was observed at temperatures above 42 °C. The colonies were yellowish, rough, slightly convex and circular with irregular margins. The cells were Gram-positive, oxidase-positive, non-motile, non-spore-forming rods and showed primary branching, which is uncommon among members of the genus Microbacterium. A phase-contrast micrograph of cells of strain S3-ET is available as Supplementary Fig. S1 in IJSEM Online. Strain S3-ET differed significantly from M. hominis and M. foliorum, its nearest phylogenetic neighbours, in terms of acid production, carbon-source utilization and substrate hydrolysis profiles as well as colony morphology. The 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 and galactose. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol and unknown polar lipids including glycolipid and phospholipid. The detailed chemotaxonomic characteristics are summarized in Table 2.
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). An extended version of the 16S rRNA gene phylogenetic tree, including all Microbacterium species with validly published names, is available as Supplementary Fig. S2 in IJSEM Online. The DNA–DNA hybridization levels between strain S3-ET and its nearest phylogenetic neighbours, M. hominis and M. foliorum, were 38·5 and 29·8 %, respectively, which confirmed that strain S3-ET could be differentiated from its nearest phylogenetic neighbours at the genomic level, as previous assumed (Stackebrandt & Goebel, 1994).
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Description of Microbacterium xylanilyticum sp. nov.
Microbacterium xylanilyticum (xy.la.ni.ly'ti.cum. N.L. n. xylanum xylan, a plant polymer; Gr. adj. lutikos able to loosen, able to dissolve; N.L. adj. lyticus -a -um dissolving; N.L. neut. adj. xylanilyticum xylan-dissolving).
Cells are non-motile, non-spore-forming rods and show primary branching. Gram-positive, oxidase-positive and catalase-positive. Good growth occurs on R2A agar, trypticase soy agar and nutrient agar at 15–37 °C, but growth is weak at 5 °C and no growth is observed at temperatures above 42 °C or on MacConkey agar. The optimal growth temperature is 25–30 °C and the optimal pH is 6–8. Growth occurs in the presence of 1, 2 and 4 % NaCl, but growth is weak in the presence of 6·5 % NaCl. Colonies are yellowish, rough, slightly convex and circular with irregular margins. Indole and H2S are not produced. The methyl-red test is negative, but the Voges–Proskauer test is positive. Nitrate is reduced, but nitrite is not reduced. Aesculin, starch and xylan are hydrolysed, but casein, cellulose and urea are not hydrolysed. According to the results from the API ZYM test, L-leucyl 2-naphthylamide, 2-naphthyl phosphate (pH 5·4), 6-bromo-2-naphthyl 
-D-galactopyranoside, 2-naphthyl -D-glucopyranoside and 2-naphthyl -L-fucopyranoside are hydrolysed, but 2-naphthyl phosphate (pH 8·5), 2-naphthyl butyrate, 2-naphthyl caprylate, 2-naphthyl myristate, L-valyl 2-naphthylamide, L-cystyl 2-naphthylamide, N-benzoyl-DL-arginine 2-naphthylamide, N-glutaryl-phenylalanine 2-naphthylamide, naphthol-AS-BI-phosphate, 2-naphthyl -D-galactopyranoside, naphthol-AS-BI--D-glucuronide, 6-bromo-2-naphthyl -D-glucopyranoside, 1-naphthyl N-acetyl--D-glucosaminide and 6-bromo-2-naphthyl -D-mannopyranoside are not hydrolysed. Acid is produced from glycerol, D-arabinose, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, mannitol, methyl -D-glucoside, amygdalin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, raffinose, starch, glycogen, D-turanose and D-xylose, but not from erythritol, L-xylose, adonitol, methyl -D-xyloside, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl -D-mannoside, N-acetylglucosamine, arbutin, inulin, xylitol, gentiobiose, 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: mannitol, D-glucose, N-acetylglucosamine, D-ribose, D-melibiose, D-sucrose, maltose, L-arabinose, DL-lactate, histidine, glycogen and L-proline. The following carbon sources are not utilized: rhamnose, salicin, inositol, L-fucose, D-sorbitol, itaconate, propionate, suberate, caprate, malonate, valerate, acetate, citrate, malate, L-alanine, 2-ketogluconate, 5-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate, 3-hydroxybenzoate and L-serine. Menaquinones MK-12, MK-11 and MK-13 are the major quinones. The fatty acid profile is composed largely of 15 : 0 anteiso (52·6 %), 16 : 0 iso (25·4 %) and 17 : 0 anteiso (11·2 %). The cell-wall peptidoglycan is of the B2 type, (L-homoserine)-D-gluglyD-Orn, with glycolyl residues. The cell-wall sugars are glucose and galactose. The polar lipids comprise diphosphatidylglycerol, phosphatidylglycerol and unknown polar lipids including glycolipid and phospholipid. The G+C content of the DNA of the type strain is 69·7 mol%.
The type strain is S3-ET (=DSM 16914T=KCTC 19079T), isolated from the biofilm of a membrane bioreactor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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