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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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7c and C18 : 1 2-OH. The DNA G+C content was 57.0 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain K107T was closely related to the genera Mycoplana, Brucella and Ochrobactrum. Strain K107T exhibited 16S rRNA gene sequence similarity values of 96.3–97.1 % with respect to the type strains of two Mycoplana species and 94.8–96.8 % with respect to members of the genera Brucella and Ochrobactrum. A phylogenetic analysis based on recA gene sequences showed that strain K107T forms a distinct phylogenetic lineage within the Alphaproteobacteria. The recA gene sequence of strain K107T showed similarity values of 84.5 % with respect to type strains of Brucella species and values of 77.6–83.1 % with respect to members of the genera Pseudochrobactrum, Ochrobactrum and Mycoplana. Strain K107T could be differentiated from phylogenetically related genera by differences in phenotypic properties and fatty acid profiles. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain K107T represents a novel genus and species, for which the name Daeguia caeni gen. nov., sp. nov. is proposed. The type strain of Daeguia caeni is strain K107T (=KCTC 12981T =CCUG 54520T).
Cellular fatty acid compositions for strain K107T and two Mycoplana type strains are presented in a supplementary table available with the online version of this paper.
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MAIN TEXT |
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, b, c, d). This study focuses on one of these isolates, designated strain K107T. Comparative 16S rRNA gene sequence analysis indicated that strain K107T is phylogenetically closely related to the family Brucellaceae within the Alphaproteobacteria. The aim of the present work was to determine the exact taxonomic position of strain K107T by using a polyphasic characterization that included determination of its phenotypic properties and detailed phylogenetic analyses based on its 16S rRNA and recA gene sequences.
Strain K107T was isolated on trypticase soy agar (TSA; Difco) at 30 °C using the standard dilution plating technique. Mycoplana dimorpha DSM 7138T and Mycoplana ramosa DSM 7292T, which were used as reference strains for fatty acid analyses, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The morphological, physiological and biochemical characteristics of strain K107T were investigated using routine cultivation on TSA at 37 °C. Cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy with cells from exponentially growing cultures. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–60 °C) was investigated on TSA. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (pH 4.5–10.5, in increments of 0.5 pH units) by the addition of HCl or Na2CO3. Growth in the absence of NaCl and at various NaCl concentrations (0.5 %, w/v, and 1.0–7.0 %, w/v, using increments of 1.0 %) was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was excluded. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and on TSA supplemented with potassium nitrate (0.1 %, w/v), both of which had been prepared anaerobically under nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). The hydrolysis of aesculin and the reduction of nitrate were studied as described previously (Lanyi, 1987). Susceptibility to antibiotics was tested on TSA plates using antibiotic discs containing the following: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; and carbenicillin, 100 µg. The utilization of various substrates, enzyme activities and other physiological and biochemical properties were tested by using the API 20E, API 20NE, API 50 CH and API ZYM systems (bioMérieux). The cells were suspended in AUX medium, according to the manufacturer's instructions, for inoculation of the API 50 CH system.
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultivation in trypticase soy broth (Difco) at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by using a PCR with two universal primers, 5'-GAGTTTGATCCTGGCTCAG-3' and 5'-AGAAAGGAGGTGATCCAGCC-3', as described previously (Yoon et al., 1998). PCR amplification of a partial recA gene was performed by using two primers, 5'-CGKCTSGTAGAGGAYAAATCGGTGGA-3' and 5'-CGRATCTGGTTGATGAAGATCACCAT-3', as described by Gaunt et al. (2001). Sequencing of the amplified 16S rRNA and recA genes and phylogenetic analysis were performed as described by Yoon et al. (2003). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. For the cellular fatty acid analysis, cell mass of strain K107T, M. dimorpha DSM 7138T and M. ramosa DSM 7292T was harvested from TSA plates after incubation for 3 days at 30 °C. The fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined using the method of Tamaoka & Komagata (1984), with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
Morphological, cultural, physiological and biochemical characteristics of strain K107T are given in the species description or are shown in Table 1. The almost-complete 16S rRNA gene sequences of strain K107T, M. dimorpha DSM 7138T and M. ramosa DSM 7292T determined in this study comprised 1441, 1440 and 1440 nt, respectively, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree constructed using the neighbour-joining algorithm, strain K107T joined the cluster comprising the type strains of M. dimorpha and M. ramosa (Fig. 1). This relationship was maintained in the tree based on the maximum-parsimony algorithm, but not in the tree based on the maximum-likelihood algorithm (Fig. 1). Strain K107T exhibited 16S rRNA gene sequence similarity values of 96.3 and 97.1 % with respect to M. dimorpha DSM 7138T and M. ramosa DSM 7292T, respectively, 96.8 % to Brucella melitensis ATCC 23456T, 94.8–96.7 % to the type strains of Ochrobactrum species and 94.7–95.1 % to the type strains of Pseudochrobactrum species. The recA gene sequence (550 nt) for strain K107T was determined in this study: it showed the highest level of similarity (84.5 %) with respect to type strains of Brucella species, the sequences of which are identical. The recA gene sequence of strain K107T had similarity values of 82.0–83.1 % with respect to the type strains of Pseudochrobactrum species, 79.1–80.7 % to the type strains of Ochrobactrum species and 77.6–77.9 % to the type strains of Mycoplana species.
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7c (66.2 %) and C18 : 1 2-OH (10.3 %) (see Supplementary Table S1 in IJSEM Online). This fatty acid profile was similar to those of the two Mycoplana species analysed in this study (Supplementary Table S1). However, the proportions of some fatty acids differed between strain K107T and the two Mycoplana species: in particularly, fatty acid C18 : 1 2-OH was present in significant amounts in strain K107T, but was not detected in either of the two Mycoplana species analysed (Supplementary Table S1). The DNA G+C content of strain K107T was 57.0 mol%.
In the 16S rRNA gene sequence analysis, strain K107T was phylogenetically most closely related to M. ramosa. However, strain K107T was distinguishable from M. dimorpha and M. ramosa on the basis of differences in phenotypic properties, DNA G+C contents and fatty acid profiles. Strain K107T was also distinguishable from members of the genera Brucella, Ochrobactrum and Pseudochrobactrum on the basis of differences in fatty acid profiles, particularly in the compositions of fatty acids cyclo-C19 : 08c and C18 : 1 2-OH (although these differences may be the result of differences in cultivation conditions and extraction procedures) (Dees et al., 1980, 1981; Tripathi et al., 2006; Kämpfer et al., 2007) (Table 1
). In the phylogenetic analysis based on recA gene sequences, strain K107T formed a phylogenetic lineage that is distinct from those of members of the Alphaproteobacteria, including Brucella, Ochrobactrum, Pseudochrobactrum and Mycoplana (Fig. 2). Accordingly, strain K107T represents a novel genus and species that is distinct from the genera Brucella, Ochrobactrum, Pseudochrobactrum and Mycoplana, for which the name Daeguia caeni gen. nov., sp. nov. is proposed.
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Cells are Gram-negative, non-spore-forming rods. The predominant ubiquinone is Q-10. The major fatty acid is C18 : 17c; a significant amount of C18 : 1 2-OH is present. Exhibits 16S rRNA gene sequence similarity values of 94.7–97.1 % with respect to members of the genera Brucella, Mycoplana, Ochrobactrum and Pseudochrobactrum. The recA gene (550 nt) exhibits sequence similarity values of 77.6–84.5 % with respect to the genera Brucella, Mycoplana, Ochrobactrum and Pseudochrobactrum. The DNA G+C content of the type strain of the type species is 57.0 mol%. The type species is Daeguia caeni.
Description of Daeguia caeni sp. nov.
Daeguia caeni (cae'ni. L. gen. n. caeni of sludge).
Exhibits the following properties in addition to those given in the genus description. Cells are 0.4–0.6x0.7–2.0 µm in size. Colonies on TSA are circular, convex, smooth, glistening, greyish yellow in colour and 1.0–1.5 mm in diameter after 2 days incubation at 37 °C. The optimal temperature for growth is 30–37 °C. Growth occurs at 15 and 55 °C, but not at 10 or 56 °C. The optimal pH for growth is 7.0–8.0; growth occurs at pH 5.0 and 9.0, but not at pH 4.5 or 9.5. Growth occurs in the presence of 0–4 % (w/v) NaCl; optimal growth occurs in the presence of 0.5–1.0 % (w/v) NaCl. No anaerobic growth occurs on TSA or on TSA supplemented with nitrate. Hypoxanthine is hydrolysed, but aesculin, casein, gelatin, starch, tyrosine, xanthine, urea and Tweens 20, 40, 60 and 80 are not. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. H2S and indole are not produced. Adonitol, N-acetylglucosamine and phenylacetate are assimilated and adipate is weakly assimilated, but erythritol, D-arabinose, L-xylose, methyl β-D-xyloside, sorbose, dulcitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, arbutin, aesculin, salicin, cellobiose, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate, 5-ketogluconate, caprate, citrate and malate are not assimilated. Alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, β-galactosidase, β-glucuronidase, -glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase are absent. Susceptible to carbenicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, streptomycin and tetracycline, but not to ampicillin, lincomycin, oleandomycin, penicillin G or polymyxin B. Other phenotypic characteristics are given in Table 1.
The type strain, K107T (=KCTC 12981T =CCUG 54520T), was isolated from sludge from a textile dye works in Daegu, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
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Yoon, J.-H., Kang, S.-J., Kim, W. & Oh, T.-K. (2007a). Pigmentiphaga daeguensis sp. nov., isolated from wastewater of a dye works, and emended description of the genus Pigmentiphaga. Int J Syst Evol Microbiol 57, 1188–1191.
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Zurdo-Pi
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