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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
Tae-Kwang Oh
otk{at}kribb.re.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH as the major fatty acids. The DNA G+C content was 49·0 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DS-44T belongs to the genus Algoriphagus of the phylum Bacteroidetes. Similarity values between the 16S rRNA gene sequences of strain DS-44T and those of the type strains of recognized Algoriphagus species were in the range 93·8–95·7 %, making it possible to categorize strain DS-44T as a species that is separate from previously described Algoriphagus species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain DS-44T (=KCTC 12545T=CIP 108837T) was classified in the genus Algoriphagus as the type strain of a novel species, for which the name Algoriphagus terrigena sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DS-44T is DQ178979.
Details of the fatty acid compositions of Algoriphagus species are available in a supplementary table in IJSEM Online.
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. At the time of writing, the genus consists of eight species with validly published names: Algoriphagus ratkowskyi (the type species; Bowman et al., 2003), A. aquimarinus, A. chordae and A. winogradskyi (Nedashkovskaya et al., 2004), A. halophilus (Yi & Chun, 2004; Nedashkovskaya et al., 2004), A. antarcticus (Van Trappen et al., 2004), A. yeomjeoni (Yoon et al., 2005a) and A. locisalis (Yoon et al., 2005b). Algoriphagus species have been isolated from marine environments, Antarctic lakes and salterns (Bowman et al., 2003; Nedashkovskaya et al., 2004; Van Trappen et al., 2004; Yoon et al., 2005a, b). In this study, we report on the taxonomic characterization of an Algoriphagus-like bacterial strain, DS-44T, which was isolated from soil from Dokdo in Korea.
Strain DS-44T was isolated by using the standard dilution plating technique on 10x diluted nutrient agar (Difco) with distilled water at 25 °C. The morphological, physiological and biochemical characteristics of strain DS-44T were investigated using routine cultivation on marine agar 2216 (MA; Difco) at 25 °C. The cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was determined using a transmission electron microscope with cells from exponentially growing cultures. Gliding motility was investigated as described by Bowman (2000). The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. The pH range for growth was determined in marine broth 2216 (MB; Difco) adjusted to various pH values (pH 4·5–10·5 at increments of 0·5 pH units). Before sterilization, the pH was adjusted to various levels by the addition of HCl or Na2CO3. Growth in the absence of NaCl was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations [0·5 % (w/v) and 1·0–10·0 % (w/v) at increments of 1·0 %] was investigated in MB or trypticase soy broth (Difco). Growth at various temperatures (4–40 °C) was measured on MA. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically under nitrogen. Catalase and oxidase activities and the hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, L-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). H2S production was tested as described previously (Bruns et al., 2001). The presence of flexirubin-type pigments was investigated as described by Reichenbach (1992). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of substrates as sole carbon and energy sources was tested as described by Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Susceptibility to antibiotics was tested on MA plates using antibiotic discs containing the following concentrations: 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; carbenicillin, 100 µg. Other physiological and biochemical tests were performed with the API 20E and API ZYM systems (bioMérieux).
Cell biomass of strain DS-44T for DNA extraction and for isoprenoid quinone analysis was obtained by cultivation for 3 days in MB at 25 °C. Chromosomal DNA was isolated and purified according to a method described previously (Yoon et al., 1996), except 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, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analyses 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 with a YMC ODS-A (250x4·6 mm) column. For fatty acid methyl ester analysis, cell mass of strain DS-44T was harvested from MA plates after incubation for 7 days at 25 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
The morphological, cultural, physiological and biochemical characteristics of strain DS-44T are given in the species description (see below) or are shown in Table 1. The almost-complete 16S rRNA gene sequence of strain DS-44T, comprising 1479 nt (approx. 96 % of the Escherichia coli 16S rRNA gene sequence), was determined in this study. 16S rRNA gene sequence analyses showed that strain DS-44T was most closely affiliated to the genus Algoriphagus of the phylum Bacteroidetes (Fig. 1). In the neighbour-joining tree based on 16S rRNA gene sequences, strain DS-44T fell within the radiation of the cluster comprising Algoriphagus species (Fig. 1). The same tree topology was found in the trees generated with the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DS-44T exhibited 16S rRNA gene sequence similarity values of 93·8–95·7 % with respect to the eight recognized Algoriphagus species, 93·2–93·4 % with respect to Hongiella species and less than 89·8 % with respect to the other species used in the phylogenetic analysis.
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9c (8·5 %), iso-C16 : 1 H (2·5 %), anteiso-C15 : 0 (2·4 %), anteiso-C11 : 0 (1·6 %), iso-C11 : 0 (1·1 %), iso-C14 : 0 (1·0 %), iso-C15 : 1 G (1·0 %) and iso-C16 : 0 (1·0 %), summed feature 3, comprising C16 : 17c and/or iso-C15 : 0 2-OH (24·5 %), and summed feature 4, comprising iso-C17 : 1 I and/or anteiso-C17 : 1 B (2·5 %), hydroxy fatty acids iso-C17 : 0 3-OH (6·9 %) and iso-C15 : 0 3-OH (3·2 %) and unsaturated fatty acids C16 : 15c (2·8 %) and C15 : 16c (1·0 %). This fatty acid profile was similar with those of previously analysed Algoriphagus species, although there were differences in the proportions of some fatty acids, perhaps because of differences in cultivation conditions (see Supplementary Table S1 in IJSEM Online). The DNA G+C content of strain DS-44T was 49·0 mol%, which is higher than those of other Algoriphagus species (Table 1
). These chemotaxonomic properties were in agreement with the result of monothetic phylogenetic classification of strain DS-44T as a member of the genus Algoriphagus (Bowman et al., 2003; Nedashkovskaya et al., 2004; Van Trappen et al., 2004; Yoon et al., 2005a, b). Strain DS-44T was distinguishable from other Algoriphagus species by means of differences in several phenotypic properties, as shown in Table 1. The 16S rRNA gene sequence similarity data were sufficient to allocate strain DS-44T to a species that is separate from the recognized Algoriphagus species (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-44T should be placed in the genus Algoriphagus as a member of a novel species, for which the name Algoriphagus terrigena sp. nov. is proposed.
Description of Algoriphagus terrigena sp. nov.
Algoriphagus terrigena (ter.ri.ge'na. L. masc. or fem. n. terrigena child of the earth, referring to the isolation of the type strain from soil).
Cells are Gram-negative, non-spore-forming, non-flagellated short rods or rods (0·4–0·6x0·8–2·5 µm); a few cells greater than 50 µm in length are also observed. Colonies on MA are circular, convex, smooth, glistening, light orange in colour and 1·0–2·0 mm in diameter after incubation for 7 days at 25 °C. Optimal growth occurs at 25 °C; growth occurs at 10 and 36 °C, but not at 4 or 37 °C. Optimal pH for growth is 6·5–7·5; growth occurs at pH 5·5, but not at pH 5·0. Optimal growth occurs in the presence of 2 % (w/v) NaCl; growth does not occur in the absence of NaCl or in the presence of >7 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Aesculin and Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine, xanthine, L-tyrosine and urea are not. H2S is not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase are present, -mannosidase is weakly present, but lipase (C14), cystine arylamidase, -galactosidase, -glucuronidase and -fucosidase are absent. D-Cellobiose, D-fructose, D-galactose, maltose, sucrose, D-trehalose, D-xylose and salicin are utilized as sole carbon and energy sources, but acetate, benzoate, formate, L-glutamate, pyruvate and succinate are not utilized. Acid is produced from D-mannose, D-raffinose and D-trehalose, weakly produced from D-fructose, D-melezitose and D-ribose, but not produced from myo-inositol, D-mannitol or D-sorbitol. Susceptible to chloramphenicol and novobiocin, but not to cephalothin. The predominant menaquinone is MK-7. The major fatty acids (>10 % of total fatty acids) are iso-C15 : 0 and C16 : 17c and/or iso-C15 : 0 2-OH. The DNA G+C content is 49·0 mol% (HPLC). Other phenotypic properties are shown in Table 1.
The type strain, DS-44T (=KCTC 12545T=CIP 108837T), was isolated from soil from Dokdo, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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