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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 National University of Uzbekistan, Students Town, Tashkent, 700-174, Uzbekistan
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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7c, C16 : 0, C15 : 0 and C17 : 0) and 16S rRNA gene sequence analysis supported the affiliation of strain Slu-01T to the genus Phenylobacterium. However, DNA–DNA hybridization data and phenotypic properties showed that strain Slu-01T could be distinguished from the two other Phenylobacterium species with validly published names. Thus, it is suggested that strain Slu-01T represents a novel species of the genus Phenylobacterium, for which the name Phenylobacterium koreense is proposed. The type strain is Slu-01T (=KCTC 12206T=IAM 15119T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Slu-01T is AB166881.
A table comparing the cellular fatty acid profiles of strain Slu-01T, Phenylobacterium immobile and Phenylobacterium lituiforme is available as supplementary material in IJSEM Online.
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and at present comprises only two species, Phenylobacterium immobile DSM 1986T (Lingens et al., 1985) and Phenylobacterium lituiforme DSM 14363T (Kanso & Patel, 2004). Kanso & Patel (2004) proposed an emendment to the description of the genus Phenylobacterium which states that members of the genus Phenylobacterium are Gram-negative, non-spore-forming, straight to slightly curved rods, coccobacilli or cocci measuring 0·7–1·0x1·0–2·0 µm and occur singly, in pairs or short chains. Species may be strict aerobes or facultative anaerobes and may be motile or non-motile. Members of the genus grow on L-phenylalanine. Based on the 16S rRNA gene sequence analysis, members of this genus form a monophyletic group within the order Caulobacterales, subclass 
-Proteobacteria, class Proteobacteria. Strain Slu-01T was isolated from activated sludge from a wastewater treatment plant in Daejeon, South Korea. In this study, a polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences and analysis of chemotaxonomic and phenotypic properties, was adopted to determine the precise taxonomic position of Slu-01T. The results indicate that Slu-01T can be assigned as a member of the genus Phenylobacterium, but the strain can be clearly distinguished from the existing Phenylobacterium species with validly published names. We propose Slu-01T as the type strain of a novel species, namely Phenylobacterium koreense sp. nov.
The serial dilution method was used on R2A (Difco) agar plates to isolate aerobic bacteria from activated sludge. Slu-01T was one of the dominant isolates appearing on the plates in aerobic conditions. Strain Slu-01T was deposited in the Korean Collection for Type Cultures (KCTC 12206T) and the IAM Culture Collection of the University of Tokyo (IAM 15119T).
The Gram reaction was performed by the non-staining method as described by Buck (1982). The morphology of cells grown for 3 days at 30 °C on R2A agar was observed under a Nikon light microscope at x1000 magnification. Catalase and oxidase tests were performed according to Cappuccino & Sherman (2002). Substrate utilization as a sole carbon source and some physiological characteristics were determined using the API 32GN, API 20E and API 20NE galleries according to the manufacturer's instructions (bioMérieux). Degradation of DNA (using Difco DNA agar, supplemented with 0·01 % toluidine blue, Merck) and degradation of casein, chitin, starch, L-tyrosine and skimmed milk (Atlas, 1993; Lewin & Lounsbery, 1969) reactions were read after 5 days.
Hydrolysis of carboxymethylcellulose was tested as described by Ten et al. (2004). Growth tests with L-phenylalanine (1·0 g l–1), chloridazon (1·0 g l–1) and antipyrin (1·0 g l–1) as the sole carbon source were determined in minimal medium B (Lingens et al., 1985) with vitamin B12 (0·03 mg l–1) at 30 °C. Growth at different temperatures and pH was assessed after 5 days incubation. Salt tolerance was tested in R2A broth medium supplemented with 1–10 % (w/v) NaCl after 7 days incubation. Duplicate antibiotic-sensitivity tests were performed using filter paper disks containing the following: streptomycin (5, 10 and 15 µg ml–1) (Mast Diagnostics), tetracycline (5, 10 and 15 µg ml–1), kanamycin (1·0, 1·5 and 2·0 mg ml–1), ampicillin (30, 50 and 60 µg ml–1) (Sigma), chloramphenicol (10, 20 and 30 µg ml–1), carbenicillin (100, 200 and 300 µg ml–1), rifampicin (50, 100 and 150 µg ml–1) and hygromycin (100, 200 and 300 µg ml–1). Disks were placed on R2A plates spread with Slu-01T culture and incubated at 30 °C for 7 days. Higher antibiotic concentrations were tested if antibiotic resistance was noted. The physiological and biochemical characteristics of strain Slu-01T and related type strains are summarized in Table 1.
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. The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed using the CLUSTAL_X program (Thompson et al., 1997). Gaps were edited using the BioEdit program (Hall, 1999). The Kimura two-parameter model (Kimura, 1983) was used to calculate evolutionary distances. A phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the MEGA2 program (Kumar et al., 2001) with bootstrap values based on 1000 replications (Felsenstein, 1985).
The G+C content of chromosomal DNA was determined using a reverse-phase HPLC method (Mesbah et al., 1989). Respiratory quinones were extracted from cells grown on R2A Broth (Difco) and analysed as described by Komagata & Suzuki (1987) using reverse-phase HPLC. Cellular fatty acids were determined from cultures grown on trypticase soy agar (TSA; Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol for the Sherlock Microbial Identification System (MIDI). Fatty acids were analysed by GC (6890; Hewlett Packard) and identified by the Microbial Identification software package (Sasser, 1990).
Strain Slu-01T was Gram-negative. Cells were rods and coccal rods (0·7–1·0x1·0–2·0 µm), slightly curved and occurred both singly and in pairs. No filaments were found, even in old cultures. No flagella movement was observed by light microscopy. Colonies grown on R2A agar plates for 3 days were smooth, circular, convex, colourless and 0·7–1·2 mm in diameter. When grown on medium C (Lingens et al., 1985) supplemented with L-phenylalanine (0·1 %) and vitamin B12 (0·03 mg l–1), Slu-01T formed a 2 mm colony after 4 days at 30 °C. Strain Slu-01T could not grow with chloridazon and antipyrin as the single carbon source. Spores were never observed and cells were heat-sensitive.
On R2A agar, the optimal growth temperature for Slu-01T was 30 °C. Strain Slu-01T was able to grow at 37 °C, but not at 4 or 45 °C. The pH growth range was between pH 6·5 and 8·5, with an optimum between pH 7 and 8. Growth occurred in the absence of NaCl and in the presence of 2·0 % (w/v) NaCl, but not 4 % (w/v) NaCl. In the presence of NaCl, most of the cells became long aberrant forms (Fig. 1). Strain Slu-01T was resistant to 500 µg ml–1 ampicillin, 50 µg ml–1 tetracycline, 50 µg ml–1 streptomycin, 2·0 mg ml–1 kanamycin, 300 µg ml–1 carbenicillin, 500 µg ml–1 hygromycin and 100 µg ml–1 rifampicin. It has been shown that it should be possible to identify species on the basis of susceptibility tests and that susceptibilities to certain antibiotics reflect adaptation to certain environmental conditions (Reva et al., 1995). The fact that strain Slu-01T exhibited resistance to antibiotics reflects its environmental habitat of activated sludge.
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The 16S rRNA gene sequence of strain Slu-01T was a continuous stretch of 1416 bp. Sequence similarity calculations after a neighbour-joining analysis indicated that the closest relatives of strain Slu-01T were P. immobile (96·6 %) and P. lituiforme (96·5 %). A low level of similarity (94 %) was found with the genera Caulobacter (Abraham et al., 1999) and Brevundimonas (Fig. 2). In order to confirm whether Slu-01T represented a novel species, DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989). An FLX 800 Microplate Fluorescence Reader (Bio-Tek) was employed for fluorescence measurements. The hybridization temperature was 50 °C and reciprocal experiments were performed for P. immobile DSM 1986T and Slu-01T. The DNA–DNA relatedness level between P. immobile DSM 1986T and Slu-01T was 15 %, which demonstrated that strain Slu-01T belonged to a distinct genomic species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Thus, based on the 16S rRNA gene sequence analyses and DNA–DNA hybridization results, a novel taxon could be detected.
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7c (23·5 %), C16 : 0 (10·4 %), C15 : 0 (9·6 %), C17 : 0 (8·7 %), C18 : 0 (7·3 %), summed feature 4 (C16 : 17c/C15 : 0 iso 2-OH, 5·7 %), C18 : 1 2-OH (4·9 %), C16 : 111c (4·4 %), C12 : 0 (4·0 %), C17 : 16c (3·7 %) and C12 : 1 3-OH (3·4 %). Although the major fatty acids of strain Slu-01T were very similar to those of type strains of species from the genus Phenylobacterium, some fatty acids, such as C11 : 0, C16 : 111c, C17 : 19c and C18 : 1 2-OH, were only present in strain Slu-01T (see Supplementary Table S1 in IJSEM Online).
Strain Slu-01T, being closely related to species of the genus Phenylobacterium, was able to metabolize the aromatic amino acid L-phenylalanine for growth and had a DNA G+C content of 68·1 mol%, within the range of 67·3–68·4 mol% for the genus Phenylobacterium (Lingens et al., 1985). Other characteristics of strain Slu-01T, such as temperature and pH requirements, colony colour and size, cell size and shape and ubiquinone (Q-10) profiles (Table 1), showed that it belonged to the genus Phenylobacterium. However, the low DNA–DNA relatedness with P. immobile DSM 1986T and other characteristics, such as high resistance to antibiotics, no metabolism of chloridazon and antipyrin as the sole carbon source, tolerance of NaCl (2 %) and fast growth on R2A agar and nutrient agar, support the recognition of a novel species within the genus Phenylobacterium. It is concluded that strain Slu-01 represents a novel species for which the name Phenylobacterium koreense sp. nov. is proposed.
Description of Phenylobacterium koreense sp. nov.
Phenylobacterium koreense (ko.re.en'se. N.L. neut. adj. koreense of Korea, from where the novel organism was isolated).
Cells are aerobic, Gram-negative, rod-shaped, non-spore-forming, non-motile, 0·7–1·0 µm widex1·0–2·0 µm long and slightly curved. Cells occur singly and in pairs. Cells do not make filaments. Colonies on R2A and complex medium are circular, convex, colourless and 0·7–1·2 mm after 3 days. The temperature range for growth is 20–37 °C, with an optimum temperature of 28–30 °C. No growth occurs at 4 or 45 °C. The pH growth range is between pH 6·5 and 8·5, with an optimum of between pH 7 and 8. Growth occurs in the absence of NaCl and in the presence of 2·0 % (w/v) NaCl, but not at 4 % (w/v) NaCl. In the presence of NaCl, most of the cells become long aberrant forms. Catalase- and oxidase-positive. H2S is not produced. Nitrate is reduced. Tests for urease, 
-glucosidase and -galactosidase activity are negative. No utilization of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, gelatinase or citrate. Does not produce any acid or gas from glucose. Casein, xylan, chitin, tyrosine, CM-cellulose, gelatin and starch are not degraded. DNA is degraded. The following compounds are utilized as sole carbon sources: L-phenylalanine, acetate, suberate, L-proline, adipate, 4-hydroxy-benzoate, 3-hydroxy-butyrate, valerate and caprate. The following compounds are not utilized as sole carbon sources: chloridazon, antipyrin, glucose, mannose, N-acetylglucosamine, maltose, propionate, L-arabinose, mannitol, gluconate, malate, citrate, phenylacetate, salicin, D-melibiose, L-fucose, D-sorbitol, histidine, 2-ketogluconate, 4-hydroxy-butyrate, rhamnose, D-ribose, inositol, D-sucrose, itaconate, malonate, lactate, L-alanine, 5-ketogluconate, glycogen and L-serine. Acetoin is produced. The species is resistant to 500 µg ml–1 ampicillin, 50 µg ml–1 tetracycline, 50 µg ml–1 streptomycin, 2·0 mg ml–1 kanamycin, 300 µg ml–1 chloramphenicol, 300 µg ml–1 carbenicillin, 500 µg ml–1 hygromycin and 100 µg ml–1 rifampicin. The major cellular fatty acids are C18 : 17c (23·5 %), C16 : 0 (10·4 %), C15 : 0 (9·6 %), C17 : 0 (8·7 %), C18 : 0 (7·3 %), summed feature 4 (C16 : 17c/C15 : 0 iso 2-OH; 5·7 %), C18 : 1 2-OH (4·9 %), C16 : 111c (4·4 %), C12 : 0 (4·0 %), C17 : 16c (3·7 %) and C12 : 1 3-OH (3·4 %). The genomic DNA G+C content of the type strain is 68·1 mol% (as determined by HPLC).
The type strain, Slu-01T (=KCTC 12206T=IAM 15119T), was isolated from activated sludge from a wastewater treatment plant in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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