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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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A table detailing the cellular fatty acid contents of strains R27T, R44, R45 and R47 is available with the online version of this paper.
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, 2006a; Kim et al., 2005; Yoon et al., 2005; Lim et al., 2006). In recent years, in an attempt to investigate the microbial community of oil-contaminated tidal flat sediment in Kwangyang, Korea, many bacterial strains have been isolated and characterized taxonomically (Jung et al., 2006b). Four of these strains, designated R27T, R44, R45 and R47, which contain LL-diaminopimelic acid in the cell-wall peptidoglycan, are the subject of this study. Comparative 16S rRNA gene sequence analysis indicated that the four strains are phylogenetically closely related to the family Propionibacteriaceae. Accordingly, the aim of the present work was to determine the exact taxonomic positions of strains R27T, R44, R45 and R47 using a polyphasic approach that included phenotypic properties, detailed phylogenetic analysis based on 16S rRNA gene sequences, and genetic relatedness.
Oil-contaminated tidal flat sediment collected from Kwangyang, Korea, was used as the source for the isolation of bacterial strains. Sediment samples (each 0.5 mg) were inoculated in 100 ml Bushnell-Haas broth (Difco) that contained 2 % (w/v) diesel oil. Cultures were incubated at 30 °C on a horizontal shaker at 150 r.p.m. Strains R27T, R44, R45 and R47 were isolated from an enrichment culture with diesel oil-degradation activity by means of dilution plating on R2A agar (Difco). To investigate their morphological, physiological and biochemical characteristics, the four novel strains were routinely cultivated on R2A agar at 30 °C. Growth at various temperatures from 4 to 50 °C was measured on R2A agar. The pH range for growth was determined in R2A broth [prepared according to the formula of the Difco medium without agar and supplemented with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 0.1 % (v/v) trace element solution (Drews, 1983)] which was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on R2A agar and on R2A agar supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Cell morphology and flagellation were examined by light microscopy (E600; Nikon) and transmission electron microscopy (TEM) by using cells grown on R2A agar. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Catalase and oxidase activities and hydrolysis of casein, starch, hypoxanthine, xanthine, tyrosine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and urea and nitrate reduction were studied as described by Lanyi (1987). Acid production from carbohydrates was determined according to the method described by Leifson (1963). Utilization of various substrates for growth was assessed according to Yurkov et al. (1994). The API ZYM system (bioMérieux) was used to determine enzyme activity. Susceptibility to antibiotics was tested on R2A agar plates using discs containing the following concentrations of antibiotic: 10 µg ampicillin; 100 µg carbenicillin; 30 µg cephalothin; 100 µg chloramphenicol; 30 µg gentamicin; 15 µg lincomycin; 30 µg kanamycin; 30 µg neomycin; 5 µg novobiocin; 15 µg oleandomycin; 20 U penicillin G; 100 U polymyxin B; 50 µg streptomycin and 30 µg tetracycline.
Strains R27T, R44, R45 and R47 were cultivated for 3 days in R2A broth prepared according to the formula of the Difco medium without agar and supplemented with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 0.1 % trace element solution (Drews, 1983) at 30 °C to obtain the cell biomass required for DNA extraction and for isoprenoid quinone and cell-wall analyses. Isoprenoid quinones were analysed as described previously (Komagata & Suzuki, 1987) using reverse-phase HPLC. The isomer type of the diamino acid of the cell wall was analysed according to the method of Komagata & Suzuki (1987). For fatty acid methyl ester analysis, cell mass of strains R27T, R44, R45 and R47 was harvested from R2A agar plates after cultivation for 3 days at 30 °C. Fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was extracted and purified by the procedure described previously (Yoon et al., 1996). The DNA G+C content was determined using the method of Tamaoka & Komagata (1984) with a modification in which DNA was hydrolysed and the resultant nucleotides were analysed by reverse-phase HPLC. Amplification of the 16S rRNA gene was performed according to the method described previously using two universal primers (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed according to Yoon et al. (2003). DNA–DNA reassociation was determined by the microplate hybridization method (Ezaki et al., 1989) using photobiotin-labelled DNA probes.
Morphological, cultural, physiological and biochemical characteristics of strains R27T, R44, R45 and R47 are given in the genus and species descriptions (see later) or are shown in Table 1, together with those of some phylogenetically related genera. The four novel strains were identical in all of the morphological, cultural, physiological and biochemical characteristics tested in this study, except with regard to the utilization of trehalose, benzoate and pyruvate and in the activities of naphthol-AS-BI-phosphohydrolase and 
-glucuronidase. The almost complete 16S rRNA gene sequences of the four strains determined in this study were identical and comprised 1472 nucleotides. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains R27T, R44, R45 and R47 formed an independent lineage of descent within the family Propionibacteriaceae of the suborder Propionibacterineae (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, the four novel strains joined the clade comprising the genera Propionibacterium, Tessaracoccus, Brooklawnia, Propionimicrobium and Luteococcus (Fig. 1). Strains R27T, R44, R45 and R47 exhibited 16S rRNA gene sequence similarity values of less than 94.5 % with respect to other species used in the phylogenetic analysis (Fig. 1). The 16S rRNA gene sequence similarity values between the four novel strains and phylogenetically related genera were as follows: Propionibacterium (93.3–90.0 %), Tessaracoccus (93.3 %), Brooklawnia (94.1 %), Propionimicrobium (92.0 %), Luteococcus (94.0–94.5 %) and Propioniferax (92.4 %). Strains R27T, R44, R45 and R47 exhibited a mean level of DNA–DNA relatedness of 91.2–97.3 % when their DNA was used individually as labelled DNA probes, indicating that the four novel strains are members of the same genomic species (Wayne et al., 1987). In view of the combined phenotypic, phylogenetic and genetic similarities, strains R27T, R44, R45 and R47 are considered to represent members of the same species.
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. The four strains contained a tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)] as the predominant isoprenoid quinone. The predominant menaquinone type [MK-9(H4)] of the four novel strains was the same as that of members of the family Propionibacteriaceae (Tamura et al., 1994; Yokota et al., 1994; Nakamura et al., 1995; Stackebrandt et al., 2002; Collins et al., 2003; Bae et al., 2006). The cellular fatty acid profiles of strains R27T, R44, R45 and R47 are shown in Supplementary Table S1 (available with the online version of this paper). The major component was anteiso-C15 : 0 (46.4–67.9 %). Strain R47 was slightly different from the three other strains in the proportions of anteiso-C15 : 0 and C16 : 0 (see Supplementary Table S1). The DNA G+C contents of strains R27T, R44, R45 and R47 were 68.8, 69.1, 69.0 and 69.2 mol%, respectively.
Strains R27T, R44, R45 and R47 could be clearly distinguished from other members of the family Propionibacteriaceae by differences in chemotaxonomic properties (Pitcher & Collins, 1991; Yokota et al., 1994; Maszenan et al., 1999; Stackebrandt et al., 2002; Collins et al., 2003; Bae et al., 2006). The genus Tessaracoccus contains MK-9(H4) and MK-7(H4) as the predominant menaquinones (Maszenan et al., 1999). Strains R27T, R44, R45 and R47 differ from the genera Propionimicrobium and Brooklawnia and Propionibacterium freudenreichii which contain lysine or meso-diaminopimelic acid as the diamino acid in position 3 of the peptidoglycan (Table 1
). The fatty acid profiles, particularly major fatty acids, distinguish the four novel strains from some phylogenetically related genera, although there may be differences in the proportions of some fatty acids, probably due to differences in cultivation conditions and extraction procedures (Table 1). In particular, strains R27T, R44, R45 and R47 are clearly distinguished from the genus Luteococcus by differences in major fatty acids (Table 1). The novel four strains contain anteiso-C15 : 0 as the major fatty acid, whereas members of the genus Luteococcus synthesize predominantly monounsaturated fatty acids, including C17 : 1, C16 : 1, C15 : 1 or C18 : 1 (Tamura et al., 1994; Collins et al., 2000, 2003; Supplementary Table S1). Low 16S rRNA gene sequence similarity values between the four novel strains and other members of the family Propionibacteriaceae, together with differential phenotypic properties, suggest that strains R27T, R44, R45 and R47 constitute a novel genus and species within the family Propionibacteriaceae, for which the name Aestuariimicrobium kwangyangense gen. nov., sp. nov. is proposed.
Description of Aestuariimicrobium gen. nov.
Aestuariimicrobium (Aes.tu.a.ri.i.mi.cro'bi.u.m. L. n. aestuarium part of the sea coast which, during the flood-tide, is overflowed, but at ebb-tide is left covered with mud or slime, a tidal flat; N.L. n. microbium microbe; N.L. neut. n. Aestuariimicrobium a microbe isolated from tidal flat).
Cells are Gram-positive, non-flagellated, short rods or cocci (0.6–1.2x1.2–2.0 µm). The predominant menaquinone is MK-9(H4). The cell-wall peptidoglycan contains LL-diaminopimelic acid. The major cellular fatty acid (>10 % total fatty acids) is anteiso-C15 : 0. The DNA G+C content is 68.8–69.2 mol%. Phylogenetically, the genus Aestuariimicrobium is a member of the family Propionibacteriaceae within the suborder Propionibacterineae of the order Actinomycetales. The type species is Aestuariimicrobium kwangyangense.
Description of Aestuariimicrobium kwangyangense sp. nov.
Aestuariimicrobium kwangyangense (kwang.yang.en'se. N.L. neut. adj. kwangyangense pertaining to Kwangyang, Korea, from where the type strain was isolated).
Exhibits the following properties in addition to those given in the genus description. Colonies on R2A agar are circular, convex, smooth, yellow and 0.8–1.0 mm in diameter after 3 days incubation at 30 °C. Growth does not occur under anaerobic conditions on R2A agar or on R2A agar supplemented with nitrate. Catalase-positive, oxidase-negative and urease-negative. Growth occurs at 4 and 40 °C with an optimum temperature of 30 °C. Optimal pH for growth is 7.5–8.5. Aesculin, casein and Tweens 20, 40, 60 and 80 are hydrolysed, but starch, hypoxanthine, xanthine and tyrosine are not. Nitrate is reduced to nitrite. D-Glucose, D-fructose, D-galactose, D-cellobiose, D-mannose, D-xylose, sucrose, maltose and salicin are utilized as sole carbon and energy sources, but L-arabinose, acetate, citrate, succinate, L-malate, formate and L-glutamate are not utilized. Utilization of trehalose (only strain R47 is negative) and benzoate and pyruvate (only strain R44 is positive) are variable. Acid is produced from D-sorbitol, D-xylose, D-fructose, melibiose, L-arabinose, D-melezitose, D-glucose, D-galactose, L-rhamnose, D-mannose, D-cellobiose, lactose, sucrose, maltose, trehalose and D-raffinose, but not from myo-inositol or D-mannitol. Susceptible to penicillin G, chloramphenicol, ampicillin, cephalothin, novobiocin, tetracycline and carbenicillin, but not to polymyxin B, streptomycin, gentamicin, kanamycin, lincomycin, oleandomycin or neomycin. Using the API ZYM system (bioMérieux), esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, 
-galactosidase, -galactosidase, -glucosidase and -glucosidase are present, but alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Naphthol-AS-BI-phosphohydrolase and -glucuronidase activities (only type strain is negative) are variable.
The type strain, R27T (=KCTC 19182T=JCM 14204T), was isolated from oil-contaminated tidal flat sediment of Kwangyang, Korea.
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ACKNOWLEDGEMENTS |
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