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Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Guseong-dong 373-1, Yuseong-gu, Daejeon 305-701, Republic of 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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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DB5T is AY884570.
Results of two-dimensional TLC analysis of polar lipids of strain DB5T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Sutherland, 1999; Terra & Ferreira, 1994). For the production of polysaccharases, micro-organisms are usually the most convenient sources and they can be obtained from various natural environments.
During a study of the bacterial community that produces polysaccharases, a large number of novel bacterial strains were isolated (Kang et al., 2003) from soil near the Korean city of Daejeon. On the basis of 16S RNA sequence data, one of these isolates, designated strain DB5T, was found to be a member of the genus Cellulomonas in the suborder Micrococcineae and was subjected to taxonomic investigation.
The aim of this study was to determine the taxonomic position of strain DB5T by using chemotaxonomic, physiological and DNA–DNA hybridization analyses. The results provided evidence that DB5T is a representative of a novel bacterial species.
Strain DB5T was isolated using the plate technique together with an insoluble chromogenic substrate (Ten et al., 2004). After isolation, strain DB5T was cultivated by being transferred on R2A agar (Difco) every month. Cell morphology and motility were observed under a Nikon E600 light microscope (1000x magnification) using cells grown on R2A agar for 3 days at 30 °C. Growth at different temperatures and pH values was assessed after 5 days incubation. Salt tolerance was tested in R2A broth (Difco) supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth was estimated by monitoring the OD600 value. Anaerobic growth was observed in serum bottles by adding thioglycolate (1 g l–1) to R2A broth and substituting the upper air layer with nitrogen gas. Carbon-source utilization and some enzyme activities were tested by using API 20NE, API ID 32 and API 50 CH test kits (bioMérieux). Catalase activity was determined by using 3 % (v/v) H2O2, while oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine (Acros). Degradation of DNA (using DNA agar from Difco, supplemented with 0·01 % toluidine blue from Merck), degradation of casein, cellulose and starch (Atlas, 1993), degradation of lipid (Kouker & Jaeger, 1987) and degradation of xylan (Ten et al., 2004) were also investigated; reactions were read after 5 days. Duplicate antibiotic-sensitivity tests were done using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg), tetracycline (5, 10 and 15 µg), kanamycin (1·0, 1·5 and 2·0 mg) and ampicillin (20, 25 and 30 µg) (all from Sigma). Discs were placed on R2A plates spread with DB5T culture and were then incubated at 30 °C for 5 days. The physiological and biochemical characteristics of strain DB5T and related type strains are summarized in Table 1.
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. The 16S rRNA gene partial sequences were compiled using SeqMan software; the 16S rRNA gene sequences of the test strain and related taxa (obtained from GenBank) were edited using the BioEdit program (Hall, 1999). Multiple alignments were performed by using the CLUSTAL_X program (Thompson et al., 1997). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). A phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in the MEGA2 program (Kumar et al., 2001). Bootstrap analysis with 1000 replicates was performed to obtain the confidence level of the branches (Felsenstein, 1985). All 12 species in the genus Cellulomonas were included in the phylogenetic tree.
Purified cell-wall preparations were obtained as described by Schleifer & Kandler (1972). Amino acids and peptides in cell-wall hydrolysates were analysed with two-dimensional TLC on cellulose plates using solvent systems described by Schleifer & Kandler (1972) and cell-wall sugar analysis as described by Staneck & Roberts (1974). Menaquinone was extracted from cells grown on R2A broth and analysed as described by Komagata & Suzuki (1987), using reverse-phase HPLC. Cellular fatty acids were analysed in organisms grown on trypticase soy agar (Difco) for 2 days. The cellular fatty acids were saponified, methylated, extracted and identified by the Microbial Identification software package (Sasser, 1990). Polar lipids were extracted and examined by using two-dimensional TLC (Minnikin et al., 1984).
The G+C content of the chromosomal DNA was determined as described by Mesbah et al. (1989), using reverse-phase HPLC. DNA–DNA hybridization was performed fluorometrically according to the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes (Sigma) and microdilution wells (Greiner). Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA hybridization values.
The cells of strain DB5T were found to be Gram-positive, non-motile, straight rods that were 0·3–0·6 µm in diameter and 1·6–2·3 µm in length. Colonies on R2A agar were yellow and smooth with clear edges. The strain showed negative results for catalase and oxidase activity, it was aerobic or facultatively anaerobic and it could reduce nitrate and hydrolyse aesculin, DNA, xylan and starch. The strain grew at 10–37 °C but not at 4 or 45 °C; the optimum temperature was 30 °C. Growth was observed at pH values in the range 6·5–9·0, the optimum pH being from 7·0 to 8·0. Strain DB5T showed salt tolerance of 4 % (w/v) NaCl. Details of various differentiating characteristics of strain DB5T and of phylogenetically related species are shown in Table 1
; other characteristics are given in the species description.
The peptidoglycan composition of strain DB5T corresponded to type A4
; it contained L-ornithine–D-glutamic acid, which is reported for most members of the genus Cellulomonas and has been emphasized as an important feature for delineation, at genus level, in the Actinobacteria (Stackebrandt & Schumann, 2000). In the case of Cellulomonas humilata ATCC 25174T, Gledhill & Casida (1969) found that the peptidoglycan of this strain contained lysine and ornithine. However, in our study, the determination of peptidoglycan for C. humilata ATCC 25174T showed the presence of L-ornithine–D-glutamic acid, which corresponds with the results of Stackebrandt et al. (2002); furthermore, the latter composition is found in most Cellulomonas species, including Cellulomonas xylanilytica LMG 21723T. The cell-wall sugars of strain DB5T were galactose, glucose and rhamnose. Fatty acid profiles of the strain and its closest neighbours are shown in Table 2. The major fatty acids were anteiso-C15 : 0 (51·9 %), iso-C15 : 0 (13·3 %) and C16 : 0 (10·9 %). No significant differences in the fatty acid profiles were found with respect to the other Cellulomonas species. HPLC analysis of the menaquinones revealed two peaks: the main peak corresponded to MK-9(H4) and the smaller one to MK-8(H4). In addition, the major menaquinone found in members of the family Cellulomonadaceae is MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides and other unidentified phosphoglycolipids. The results of polar lipid analysis of strain DB5T are available as supplementary data in IJSEM Online.
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) and the Ribosomal Database Project (Maidak et al., 1997). The two species most closely related to strain DB5T were found to be C. xylanilytica LMG 21723T (98·0 %) and C. humilata ATCC 25174T (97·9 %). A phylogenetic tree obtained by using the neighbour-joining method shows this close relationship (Fig. 1), which was supported by a high bootstrap value based on 1000 resamplings. Furthermore, the results of DNA–DNA hybridization showed that strain DB5T has 41 % relatedness with C. xylanilytica LMG 21723T and 20 % relatedness with C. humilata ATCC 25174T. These results suggest that strain DB5T should be classified as a novel species, particularly in view of the recommendations of Wayne et al. (1987) and Stackebrandt & Goebel (1994) for species delineation. The DNA G+C content of the strain is 73·9 mol%, a value that falls within the range reported for members of the genus Cellulomonas.
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Description of Cellulomonas terrae sp. nov.
Cellulomonas terrae (ter'rae. L. gen. n. terrae of the earth).
Grows well under aerobic or facultatively anaerobic conditions at 30 °C on R2A agar. Cells are Gram-positive, straight rods and produce creamy yellow, circular, smooth colonies. No growth at 4 or 45 °C or at 4 % NaCl. Shows negative results for oxidase and catalase activity and production of urease, indole and acylamidase, and positive results for -galactosidase, -glucosidase, reduction of nitrate and hydrolysis of aesculin, DNA, starch and xylan. Can also use the following as sole carbon sources: arabinose, cellulose, fructose, galactose, gentiobiose, glycogen, glucose, inositol, 5-ketogluconate, maltose, mannose, D-melibiose, N-acetylglucosamine, rhamnose, D-sucrose, starch and xylan. Shows no growth with the following: acetate, adipate, L-alanine, caprate, citrate, L-fucose, gluconate, 3-hydroxybenzoate, 3-hydroxybutyrate, 4-hydroxybutyrate, histidine, itaconate, 2-ketogluconate, lactate, mannitol, malate, malonate, propionate, L-proline, phenylacetate, D-ribose, salicin, L-serine, D-sorbitol, suberate and valerate. Can produce acid from L-arabinose, cellobiose, fructose, galactose, gentiobiose, glycerol, glycogen, glucose, D-lyxose, maltose, mannose, melezitose, melibiose, N-acetylglucosamine, raffinose, sucrose, trehalose, D-turanose and D-xylose. It is also resistant to discs containing 30 µg ampicillin, 15 µg tetracycline, 15 µg streptomycin and 2 mg kanamycin. The predominant isoprenoid quinones are MK-9(H4) and MK-8(H4). The most abundant cellular fatty acids are anteiso-C15 : 0 (51·9 %), iso-C15 : 0 (13·3 %) and C16 : 0 (10·9 %). The G+C content of the genomic DNA of the type strain is 73·9 mol%. The peptidoglycan contains L-orn–D-Glu (type A4). The cell-wall sugars are galactose, glucose and rhamnose.
The type strain, DB5T (=KCTC 19081T=NBRC 100819T), was isolated from soil near Yusong in Daejeon City, Republic of Korea.
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ACKNOWLEDGEMENTS |
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