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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Guseong-dong 373-1, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 Department of Oriental Medicinal Materials and Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, Republic of Korea
3 School of Biology, King George VI Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
4 Mount On Science Co. Ltd, 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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-glucosidase activities, was isolated from compost at a cattle farm near Daejeon, Republic of Korea. It was a Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas, with highest sequence similarity to Cellulomonas uda DSM 20107T (98.5 %). Cell wall analysis revealed the presence of type A4, L-orn–D-Glu peptidoglycan. The cell-wall sugars detected were mannose and glucose. The predominant menaquinone was MK-9(H4); MK-8(H4) was detected in smaller quantities. The major fatty acids were anteiso-C15 : 0, C16 : 0, C14 : 0 and C18 : 0. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The results of DNA–DNA hybridization and physiological and biochemical tests clearly demonstrated that TR7-06T represents a novel species. The combined genotypic and phenotypic data show that strain TR7-06T (=KCTC 19030T=NBRC 100758T) merits description as the type strain of a novel Cellulomonas species, Cellulomonas composti sp. nov.
API ID32 test results for strain TR7-06T and related Cellulomonas strains are available with the online version of this paper.
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) and environmental (Bagnara et al., 1985; Elberson et al., 2000) significance. The genus was proposed by Bergey et al. (1923) for several organisms which were previously classified by Kellerman & McBeth (1912) as Bacillus species. Applications of chemotaxonomic, molecular systematic and numerical phenetic methods were significant in the establishment of the genus, and these methods form the basis for its differentiation from closely related genera by a combination of morphological and biochemical properties; in particular, Cellulomonas species can be differentiated from members of related genera on the basis of chemotaxonomic and phenotypic properties (Stackebrandt & Kandler, 1979).
Along with Oerskovia, Cellulomonas currently belongs to the family Cellulomonadaceae (Stackebrandt & Schumann, 2000) in the suborder Micrococcineae (Prévot, 1961; Stackebrandt et al., 1997). At the time of writing, the genus contains 14 species with validly published names.
Strain TR7-06T was isolated along with a large member of other strains during a study of the cultivable aerobic bacterial community in compost from a cattle farm near Daejeon, Republic of Korea. Strain TR7-06T was shown to be a member of the genus Cellulomonas based on partial 16S RNA gene sequence data and it was subjected to a taxonomic investigation by using chemotaxonomic, physiological and DNA–DNA hybridization analyses. The results provided evidence that TR7-06T is a representative of a novel bacterial species.
TR7-06T was one of the isolates from the compost, which was composed of cow dung and rice straw, that grew on R2A agar (Difco) plates under aerobic conditions. The strain was maintained on R2A agar; stock cultures were preserved as a suspension in glycerol (20 %, v/v) at –70 °C. Biomass for chemotaxonomic studies was prepared by cultivating the strain in shake flasks of R2A broth (Difco) at 150 r.p.m. for 3 days at 30 °C. Cultures were checked for purity, harvested by centrifugation and freeze-dried.
Cell morphology and motility were observed under a Nikon light microscope (1000x magnification) with cells grown on R2A agar for 3 days at 30 °C. The Gram reaction was conducted according to the non-staining method described by Buck (1982). Growth at different temperatures and pH was assessed after 5 days incubation on R2A agar. Salt tolerance was tested in R2A broth supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth was estimated by monitoring OD600. Anaerobic growth was observed in serum bottles by adding thioglycolate (1 g l–1) to R2A broth and replacing the upper air layer with nitrogen gas. Carbon source utilization and some enzyme activities were tested by using the API 20NE, API ID32 and API 50CH test kits (bioMérieux). Catalase activity was determined using 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. Tests for the degradation of DNA [flooding DNase agar plates (Scharlau) with 1 M HCl], casein, chitin, starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987), xylan and cellulose (Ten et al., 2004) were performed and evaluated after 5 days. Results regarding the physiological characteristics of strain TR7-06T are summarized in the species description; the data enable the novel strain to be differentiated from other Cellulomonas species, as shown in Table 1.
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. Full sequences of the 16S rRNA gene were compiled using SEQMAN software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed using the program CLUSTAL_X (Thompson et al., 1997). Gaps were edited in the program BioEdit (Hall, 1999). Evolutionary distances were calculated using Kimura's two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using a neighbour-joining method (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) using the program MEGA3 (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
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 solvent systems described by Schleifer & Kandler (1972). Cell-wall sugars were analysed as described by Staneck & Roberts (1974). Menaquinones were extracted from cells grown in R2A broth (Difco) and analysed as described by Komagata & Suzuki (1987) using reverse-phase HPLC. Cellular fatty acid profiles were determined for strains grown on TSA (Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). Fatty acids analysed by GC (Hewlett Packard 6890) were identified by the Microbial Identification software package (Sasser, 1990). Polar lipids were extracted and examined by 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 by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. 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–DNA hybridization values.
Phenotypic properties of strain TR7-06T were consistent with its classification in the genus Cellulomonas (Stackebrandt & Kandler, 1979). It was an aerobic or facultatively anaerobic, Gram-positive, rod-shaped and non-motile actinomycete that did not form mycelium. It contained L-orn–D-Glu as diamino acid in the peptidoglycan (peptidoglycan type A4) and mannose and glucose in the cell wall (cell-wall type VIII). The predominant menaquinone was MK-9(H4) and the DNA G+C content was 73.7 mol%.
The almost complete 16S rRNA gene sequence (1453 bp) obtained for strain TR7-06T was compared with sequences of representatives of the suborder Micrococcineae. High 16S rRNA gene sequence similarity was found between the tested strain and representatives of the genus Cellulomonas (94.4–98.3 %), which supports its inclusion in the genus Cellulomonas. It is evident from Fig. 1 that strain TR7-06T is most closely related to Cellulomonas uda DSM 20107T, Cellulomonas gelida DSM 20111T and Cellulomonas iranensis ATCC 700643T, supported by a moderately high bootstrap value; 16S rRNA gene sequence similarities between strain TR7-06T and these three type strains were 97.7–98.3 % (C. uda DSM 20107T showed the highest sequence similarity). However, it is evident from DNA–DNA hybridization data that strain TR7-06T and C. uda DSM 20107T should not be classified within the same species as they shared a DNA–DNA hybridization value of 10.7 %, well below the 70 % cut-off point recommended for the delineation of genomic species (Wayne et al., 1987). The organism also has a profile of phenotypic properties that distinguish it from representatives of all species of Cellulomonas with validly published names, including C. uda, the most closely related species (Table 1
). Moreover, strain TR7-06T could be differentiated easily from the three species of the genus Cellulomonas that are phylogenetically most closely related using tests for the utilization of substrates as sole carbon sources (see Supplementary Table S1 available in IJSEM Online). Unlike C. uda DSM 20107T and C. gelida DSM 20111T, strain TR7-06T could not utilize salicin or acetate.
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Description of Cellulomonas composti sp. nov.
Cellulomonas composti (N.L. n. compostum compost; N.L. gen. n. composti of compost).
Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped cells. Colonies on R2A agar plates are circular, smooth, yellow circles within 3 days at 25 °C. Optimal growth occurs at 30 °C and pH 6.8–7.5. Utilizes D-glucose, maltose and glycogen as sole carbon sources. Acid is produced aerobically and anaerobically from L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-cellobiose, D-maltose, sucrose, D-trehalose, starch, glycogen and gentiobiose. Acid is produced weakly under anaerobic conditions from D-ribose, D-lactose, inulin and D-lyxose. Hydrolyses gelatin weakly. Positive for nitrate reduction. -Glucosidase, amylase, cellulase and DNase are produced. Negative for catalase and urease. MK-9(H4) is the predominant menaquinone. The major cellular fatty acids are anteiso-C15 : 0 (44.0 %), C16 : 0 (14.6 %), C14 : 0 (9.0 %) and C18 : 0 (7.9 %). Cell wall sugars are mannose and glucose.
The type strain is TR7-06T (=KCTC 19030T=NBRC 100758T), isolated from compost from a cattle farm near Daejeon, Republic of Korea. The G+C content of the genomic DNA of the type strain is 73.7 mol% (determined by HPLC).
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ACKNOWLEDGEMENTS |
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