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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
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7c as the major fatty acid. The major polar lipids were phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 56.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DSW-18T fell within the cluster comprising Loktanella species. The levels of 16S rRNA gene sequence similarity between strain DSW-18T and the type strains of recognized Loktanella species ranged from 94.4 to 98.4 %. The DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain DSW-18T was distinguishable from recognized Loktanella species. On the basis of the phenotypic, phylogenetic and genetic data, strain DSW-18T represents a novel species of the genus Loktanella, for which the name Loktanella maricola sp. nov. is proposed. The type strain is DSW-18T (=KCTC 12863T=JCM 14564T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSW-18T is EF202613.
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with the description of three novel species, L. salsilacus, L. fryxellensis and L. vestfoldensis. Subsequently, four other Loktanella species, L. hongkongensis (Lau et al., 2004), L. agnita and L. rosea (Ivanova et al., 2005) and L. koreensis (Weon et al., 2006), have been described. Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Loktanella belongs to the Alphaproteobacteria (Van Trappen et al., 2004; Weon et al., 2006). Here we report on the taxonomic characterization of a Loktanella-like bacterial strain, DSW-18T, which was isolated from seawater off Dokdo in the East Sea, Korea.
Strain DSW-18T was isolated by using the standard dilution plating technique at 25 °C on marine agar 2216 (MA; Difco). The type strains of two Loktanella species were used as reference strains for DNA–DNA hybridization; L. rosea LMG 22534T was obtained from the Laboratorium voor Microbiologie Universiteit Gent (Ghent, Belgium) and L. koreensis KACC 11519T was obtained from the Korean Agricultural Culture Collection (Suwon, Korea). The morphological, physiological and biochemical characteristics of strain DSW-18T were investigated using routine cultivation on MA at 25 °C. Cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella 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. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations was investigated using marine broth 2216 (MB; Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–40 °C) was measured on MA. Growth on trypticase soy agar (TSA; Difco), nutrient agar (NA; Difco) and MacConkey agar (Difco) was tested at 25 °C. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were investigated as described by Lanyi (1987) with the modification that artificial seawater was used for preparation of the media. The artificial seawater contained (l–1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). H2S production was tested as described by Bruns et al. (2001). Susceptibility to antibiotics was investigated on MA plates by using antibiotic discs containing the following amounts: 100 U polymyxin B, 50 µg streptomycin, 20 U penicillin G, 100 µg chloramphenicol, 10 µg ampicillin, 30 µg cephalothin, 30 µg gentamicin, 5 µg novobiocin, 30 µg tetracycline, 30 µg kanamycin, 15 µg lincomycin, 15 µg oleandomycin, 30 µg neomycin and 100 µg carbenicillin. Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates for growth was tested as described by Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral salts solution (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Other physiological and biochemical tests were performed using the API 20E and API ZYM systems (bioMérieux). For in vivo pigment-absorption spectrum analysis, strain DSW-18T was cultivated aerobically in the dark at 25 °C in MB. The culture was washed twice by centrifugation using MOPS buffer (MOPS/NaOH, 0.01 M; KCl, 0.1 M; MgCl2, 0.001 M; pH 7.5) and disrupted by sonication with a Branson Sonifier 450. After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on a Beckman Coulter DU800 spectrophotometer.
Cell biomass for DNA extraction and for isoprenoid quinone and polar lipid analyses was obtained from cultures grown in MB at 25 °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 PCR using two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain DSW-18T was harvested from MA plates after incubation for 3 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). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and identified by using two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). The presence of phosphatidylcholine was identified by using Dragendorff's reagent (Sigma). The DNA G+C content was determined using the method of Tamaoka & Komagata (1984) with the modification that the DNA was hydrolysed using nuclease P1 (Sigma) and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by using the method of Ezaki et al. (1989) with photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded, and the means of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain DSW-18T are given in the species description (see below) or are shown in Table 1. Strain DSW-18T produced bacteriochlorophyll (BChl) a aerobically in the dark. The sonicated cell extract showed absorption maxima at approximately 476 and 864 nm, indicating the presence of carotenoid and BChl a. The almost-complete 16S rRNA gene sequence of strain DSW-18T determined in this study comprised 1419 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree constructed using the neighbour-joining algorithm, strain DSW-18T fell within the clade comprising Loktanella species, joining the type strain of L. rosea at a bootstrap resampling value of 98.7 % (Fig. 1). This tree topology was maintained in trees based on the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DSW-18T exhibited 16S rRNA gene sequence similarity values of 98.4 and 96.9 % to the type strains of L. rosea and L. koreensis, respectively, and of 94.4–95.2 % to the type strains of the other Loktanella species. The fatty acid profile of strain DSW-18T comprised (>0.5 % of total fatty acids): unsaturated fatty acid C18 : 17c (75.9 %); 11-methyl C18 : 17c (10.2 %); straight-chain fatty acids C16 : 0 (3.9 %), C18 : 0 (2.9 %) and C17 : 0 (0.7 %); hydroxy fatty acid C12 : 1 3-OH (2.9 %); and branched fatty acid iso-C17 : 0 3-OH (1.3 %). This fatty acid profile was similar to those of other recognized Loktanella species (Van Trappen et al., 2004; Lau et al., 2004; Ivanova et al., 2005; Weon et al., 2006). The predominant isoprenoid quinone detected in strain DSW-18T was ubiquinone-10 (Q-10), at a peak area ratio of approximately 95 %. The major polar lipids found in strain DSW-18T were phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol; a minor amount of phosphatidylethanolamine was also present. The DNA G+C content of strain DSW-18T was 56.8 mol%, which was lower than those of other Loktanella species (Table 1). Strain DSW-18T showed mean levels of DNA–DNA relatedness of 17 and 9 %, respectively, to the type strains of two phylogenetically related species, L. rosea LMG 22534T and L. koreensis KACC 11519T, indicating that strain DSW-18T represents a genomic species that is different from the two Loktanella species (Wayne et al., 1987). The differential phenotypic properties, together with the phylogenetic distinctiveness and DNA–DNA relatedness data, were sufficient to categorize strain DSW-18T as a member of a species that is distinct from recognized Loktanella species (Table 1) (Stackebrandt & Goebel, 1994; Wayne et al., 1987). Therefore, on the basis of the data presented, strain DSW-18T should be placed in the genus Loktanella as representing a novel species, for which the name Loktanella maricola sp. nov. is proposed.
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Cells are Gram-negative, non-motile and rod-shaped (0.3–0.6x0.8–3.0 µm). Colonies on MA are circular, slightly convex, glistening, light orange in colour and 1.0–2.0 mm in diameter after 7 days incubation at 25 °C. Growth does not occur on MacConkey agar. Growth occurs at 4 and 34 °C, but not at 35 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs in the presence of 7 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 8 % (w/v) NaCl. Anaerobic growth does not occur on MA and on MA supplemented with nitrate. Hypoxanthine and Tweens 20, 40 and 60 are hydrolysed, but tyrosine and xanthine are not. H2S and indole are not produced. BChl a is produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4) and esterase lipase (C8) are present and leucine arylamidase and acid phosphatase are weakly present, but lipase (C14), valine arylamidase, cystine arylamidase, 
-chymotrypsin, naphthol-AS-BI-phosphohydrolase, 
-glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Utilizes the following substrates as carbon and energy sources: D-cellobiose, D-galactose, D-glucose, D-fructose, acetate, citrate, succinate, L-malate and pyruvate. L-Arabinose, maltose, D-mannose, sucrose, D-trehalose, D-xylose, benzoate, formate, L-glutamate and salicin are not utilized. Acid is not produced from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose, D-trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. Sensitive to ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamicin, neomycin, novobiocin, oleandomycin, penicillin G, streptomycin and tetracycline, but not to kanamycin, lincomycin or polymyxin B. The predominant ubiquinone is Q-10. The major fatty acid is C18 : 17c. Major polar lipids are phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of the type strain is 56.8 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1
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The type strain, DSW-18T (=KCTC 12863T=JCM 14564T), was isolated from seawater off Dokdo in the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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