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1 Division of Applied Biology and Chemistry, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, South Korea
2 Department of Oriental Medicinal Material and Processing, College of Life Sciences, Kyung Hee University, Kyunggi-do, South Korea
Correspondence
Hee-Young Jung
heeyoung{at}knu.ac.kr
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ABSTRACT |
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5c; major hydroxy fatty acid, C17 : 0 iso 3-OH) supported the affiliation of strain KP01T with the genus Chitinophaga. Therefore strain KP01T represents a novel species, for which the name Chitinophaga terrae sp. nov. is proposed. The type strain is KP01T (=KCTC 12836T =LMG 24015T).
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as including strains of filamentous, chitinolytic, gliding bacteria that transform, upon ageing, into spherical bodies. Recently, Kämpfer et al. (2006) proposed five more Chitinophaga species by reclassifying [Cytophaga] arvensicola, [Flexibacter] filiformis, [Flexibacter] japonensis and [Flexibacter] sancti into the genus Chitinophaga, and by proposing Chitinophaga skermanii. At the time of writing, there are six Chitinophaga species with validly published names.
Strain KP01T was originally isolated from soil from a field in Daejeon in South Korea. This soil sample was thoroughly suspended with 50 mM phosphate buffer (pH 7.0) and the suspension was spread on one-tenth-strength R2A agar (Difco) plates after serial dilution with 50 mM phosphate buffer (pH 7.0). The plates were incubated at 30 °C for 1 week. Single colonies were purified by transferring them onto new plates and were incubated once again, on full-strength R2A or Luria–Bertani (LB) agar (Difco) plates. Purified colonies were tentatively identified on the basis of partial sequences of the 16S rRNA gene (Im et al., 2005). They were routinely cultured on LB agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
A Gram reaction was performed using the non-staining method, as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000, with cells grown for 3 days at 30 °C on LB agar. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. Carbon-source utilization and enzyme activities were tested by using API 20NE, API ID 32 GN and API ZYM test kits (bioMérieux). Growth at different temperatures (4, 15, 20, 25, 30, 37 and 42 °C) and various pH values (pH 5.0–11.0 at intervals of 0.5 pH units) was assessed after 5 days incubation. Growth on nutrient agar and trypticase soy agar (Difco) was also evaluated at 30 °C.
Extraction of genomic DNA was performed with a commercial genomic DNA-extraction kit (Solgent). PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Kim et al. (2005). 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 with the CLUSTAL_X program (Thompson et al., 1997), gaps were edited in the BioEdit program (Hall, 1999) and evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987) with the MEGA3 program (Kumar et al., 2004); bootstrap percentages were based on 1000 replications (Felsenstein, 1985).
For the measurement of the G+C content of the chromosomal DNA, genomic DNA of the novel strain was extracted and purified as described by Moore & Dowhan (1995) and enzymically degraded into nucleosides and the G+C content was determined as described by Mesbah et al. (1989) using reversed-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1, v/v). The crude n-hexane–quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by HPLC as described previously (Hiraishi et al., 1996). Cellular fatty acid profiles were determined for strains grown on trypticase soy agar at 30 °C for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids were analysed by gas chromatography (6890; Hewlett Packard) and identified using the Microbial Identification software package (Sasser, 1990).
Strain KP01T was found to comprise Gram-negative, aerobic, non-motile, non-spore-forming rods. Colonies grown on LB agar plates for 3 days were smooth, circular and yellowish. The strain grew well on nutrient agar, trypticase soy agar and LB agar. The physiological characteristics of strain KP01T are summarized in the species description, and differential characteristics are compared with those of Chitinophaga species in Table 1.
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), but the 16S rRNA gene sequence similarity with respect to the most closely related Chitinophaga species was 95.7 %, suggesting that it is different at the species level (Stackebrandt & Goebel, 1994). Strain KP01T showed the highest level of 16S rRNA gene sequence similarity with respect to C. arvensicola IAM 12650T (95.7 %), followed by Chitinophaga ginsengisegetis M1-09T (95.6 %), Chitinophaga japonensis IFO 16041T (93.3 %), Chitinophaga sancti IFO 15057T (92.7 %), Chitinophaga filiformis IFO 15056T (91.9 %), Chitinophaga ginsengisoli Gsoil 052T (91.9 %), C. skermanii SG-1BT (91.7 %), C. pinensis ACM 2034T (90.3 %) and Terrimonas lutea 14990YT (90.4 %). These values (<97 %) were low enough, according to Stackebrandt & Goebel (1994), to justify the placement of strain KP01T as a novel species within the genus Chitinophaga.
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). The major fatty acids of the strain were C15 : 0 iso, C16 : 15c and hydroxy fatty acid C17 : 0 iso 3-OH, as shown in Table 2; this profile is consistent with those of known Chitinophaga species (Kämpfer et al., 2006). Minor amounts of saturated fatty acids C14 : 0 (0.9 %), C15 : 0 iso 3-OH (3.4 %), C16 : 0 3-OH (3.0 %), C17 : 0 iso (1.0 %) and summed features 3 (C16 : 17c/C15 : 0 iso 2-OH) (3.0 %) and 4 (C17 : 1 iso I /C17 : 1 anteiso B) (1.0 %) were present.
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Description of Chitinophaga terrae sp. nov.
Chitinophaga terrae (ter'rae. L. gen. n. terrae of the earth).
Cells are Gram-negative, aerobic, non-motile rods that are 0.3–0.5 µm in width and 0.6–0.8 µm in length after 3 days culture on R2A agar. Colonies grown on LB agar for 3 days are smooth, circular, convex and yellowish. Grows at 15–42 °C and at pH 6.0–9.0, but grows slowly at 42 °C and pH 9.0. Can reduce nitrate to nitrate but not to nitrogen gas. Enzyme activities, substrate assimilation and other physiological characteristics are indicated in Table 1
. Produces N-acetyl-
-glucosaminidase, acid phosphatase, alkaline phosphatase, 
-chymotrypsin, cystine arylamidase, esterase (C4), esterase (C8), -fucosidase, -galactosidase, -glucosidase, -galactosidase, -glucosidase, leucine arylamidase, -mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase. Does not produce arginine dihydrolase, -glucuronidase, lipase (C14), protease (gelatin hydrolysis) or urease. Assimilates L-arabinose, L-fucose, D-glucose, maltose, D-mannose, D-melibiose, L-rhamnose, sucrose, N-acetyl-D-glucosamine and salicin. Does not assimilate acetate, adipate, caprate, citrate, gluconate, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxybutyrate, itaconate, 2-ketogluconate, 5-ketogluconate, lactate, L-malate, malonate, phenylacetate, propionate, suberate, n-valerate, D-ribose, myo-inositol, D-mannitol, D-sorbitol, L-alanine, L-histidine, L-proline, L-serine or glycogen. MK-7 is the predominant menaquinone and C15 : 0 iso, C16 : 15c and C17 : 0 iso 3-OH are the predominant cellular fatty acids. The G+C content of the genomic DNA of the type strain is 46.3 mol%.
The type strain, KP01T (=KCTC 12836T=LMG 24015T), was isolated from soil from a field near Daejeon, South Korea.
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REFERENCES |
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Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hiraishi, A., Ueda, Y., Ishihara, J. & Mori, T. (1996). Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection. J Gen Appl Microbiol 42, 457–469.[CrossRef]
Im, W.-T., Jung, H.-M., Cui, Y.-S., Liu, Q.-M., Zhang, S.-L. & Lee, S.-T. (2005). Cultivation of the three hundreds of bacterial species from soil of a ginseng field and mining the novel lineage bacteria. In Proceedings of the International Meeting of the Federation of Korean Microbiological Societies, abstract A035, p. 169. Seoul: Federation of Korean Microbiological Societies.
Kämpfer, P., Young, C. C., Sridhar, K. R., Arun, A. B., Lai, W. A., Shen, F. T. & Rekha, P. D. (2006). Transfer of [Flexibacter] sancti, [Flexibacter] filiformis, [Flexibacter] japonensis, and [Cytophaga] arvensicola to the genus Chitinophaga and description of Chitinophaga skermanii sp. nov. Int J Syst Evol Microbiol 56, 2223–2228.
Kim, M. K., Im, W.-T., Ohta, H., Lee, M. & Lee, S.-T. (2005). Sphingopyxis granuli sp. nov., a -glucosidase-producing bacterium in the family Sphingomonadaceae in -4 subclass of the Proteobacteria. J Microbiol 43, 152–157.[Medline]
Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Moore, D. D. & Dowhan, D. (1995). Preparation and analysis of DNA. In Current Protocols in Molecular Biology, pp. 2–11. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley.
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Sangkhobol, V. & Skerman, V. B. D. (1981). Chitinophaga, a new genus of chitinolytic myxobacteria. Int J Syst Bacteriol 31, 285–293.
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
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