DOI: 10.15468/2kxynt
title: Colwellia mytili sp. nov., isolated from mussel Mytilus edulis

description: This dataset contains the digitized treatments in Plazi based on the original journal article Kim, Young-Ok, Park, In-Suk, Park, Sooyeon, Nam, Bo-Hye, Jung, Yong-Taek, Kim, Dong-Gyun, Yoon, Jung-Hoon (2017): Colwellia mytili sp. nov., isolated from mussel Mytilus edulis. International Journal of Systematic and Evolutionary Microbiology 67 (1): 31-36, DOI: 10.1099/ijsem.0.001564, URL: http://dx.doi.org/10.1099/ijsem.0.001564AbstractA Gram-stain-negative, aerobic, motile and rod-shaped bacterial strain, designated RA2-7 T, was isolated from a mussel (Mytilus edulis) collected from the South Sea, South Korea, and subjected to a taxonomic study using a polyphasic approach. Strain RA2-7 T grew optimally at 20 Ǫ C, at pH 7.0–8.0 and in the presence of 2.0–3.0 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain RA2-7 T belonged to the genus Colwellia. Strain RA2-7 T exhibited 16S rRNA gene sequence similarity values of 98.3, 98.0 and 97.5 % to the type strains of Colwellia sediminilitoris, Colwellia aestuarii and Colwellia polaris, respectively, and of 94.5–96.5 % to the type strains of the other species of the genus Colwellia. Strain RA2-7 T contained Q-8 as the predominant ubiquinone and summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c) and C 16: 0 as the major fatty acids. The major polar lipids detected in strain RA2-7 T were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain RA2-7 T was 39.0±0.04 mol% and its DNA–DNA relatedness values with the type strains of C. sediminilitoris, C. aestuarii and C. polaris were 14–19 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain RA2-7 T is separated from recognized species of the genus Colwellia. On the basis of the data presented, strain RA2-7 T is considered to represent a novel species of the genus Colwellia, for which the name Colwellia mytili sp. nov. is proposed. The type strain is RA2-7 T (= KCTC 52417 T = NBRC 112381 T).During a screening of novel bacteria from mussels (Mytilus edulis) collected from the South Sea, South Korea, many novel bacterial strains have been isolated and characterized taxonomically. One of these isolates, a Colwellia -like bacterial strain (designated RA2-7 T), is described in this study. The genus Colwellia was proposed by Deming et al. [1] with the reclassification of ‘ Vibrio psychroerythrus ’ as Colwellia psychrerythraea (type species). Subsequently, fourteen further species of the genus Colwellia with validly published names have been described (www.bacterio.net/colwellia. html; [2, 3]). Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Colwellia falls within the class Gammaproteobacteria [4, 5]. Members of the genus Colwellia have been isolated from marine environments and marine organisms, including seawater, marine sediment, starfish and sea squirt [1, 3, 4, 6 – 10]. The aim of the present work was to determine the exact taxonomic position of strain RA2-7 T by using a polyphasic characterization that included the determination of chemotaxonomic and other phenotypic properties, a detailed phylogenetic investigation based on 16S rRNA gene sequences, and DNA–DNA hybridization.A mussel (Mytilus edulis) was collected from Cheongsan island in the South Sea, South Korea, and its inner body was used as a source for the isolation of bacterial strains. Strain RA2-7 T was isolated by the standard dilution plating technique at 20 Ǫ C on a mixture (1: 1, v/v) of R2A agar (BD Difco) and marine agar 2216 (MA; BD Difco), and cultivated routinely at 20 Ǫ C on MA. Colwellia aestuarii SMK-10 T, Colwellia polaris JCM 13952 T and Colwellia chukchiensis LMG 25329 T, which were used as reference strains for phenotypic characterization and polar lipid analyses, were obtained from our laboratory [6], the Japan Collection of Microorganisms (JCM; Japan) and the Laboratorium voor Microbiologie Universiteit Gent (LMG; Belgium), respectively. DNAs of Colwellia sediminilitoris YSM-23 T, Colwellia aestuarii SMK-10 T and Colwellia polaris JCM 13952 T for DNA–DNA hybridization were obtained from the study of Park et al. [3]. The cell morphology, Gram reaction, pH range for growth and anaerobic growth, requirement for Mg 2+ ions and hydrolysis of gelatin and urea were determined as described by Park et al. [11]. Growth at 4, 10, 15, 20, 25, 30 and 35 Ǫ C was measured on MA to determine the optimal temperature and temperature range for growth.Author affiliations: 1 Biotechnology Research Division, National Institute of Fisheries Science (NIFS), Gijang, Busan 619-705, Republic of Korea; 2 Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea.*Correspondence: Jung-Hoon Yoon, jhyoon69@skku.eduKeywords: Mussel; Mytilus edulis; Polyphasic taxonomy; Novel species; Colwellia mytili sp. nov.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RA2-7 T is KX450475.Two supplementary figures are available with the online Supplementary Material.Growth at various concentrations of NaCl (0, 0.5 and 1.0– 7.0 %, at increments of 1.0 %) was investigated by supplementing appropriate concentrations of NaCl in marine broth 2216 (MB) prepared according to the formula of the BD Difco medium except that NaCl was excluded. Catalase and oxidase activities were determined as described by Ĺanyí [12]. Hydrolysis of casein, starch, hypoxanthine, L-tyrosine and xanthine was investigated on MA using the substrate concentrations described by Barrow and Feltham [13]. Hydrolysis of aesculin and Tween 80, and nitrate reduction were investigated as described previously [12] with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l ‒1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl 2. 6H 2 O, 5.94 g MgSO 4. 7H 2 O and 1.3 g CaCl 2. 2H 2 O [14]. Utilization of various substrates for growth was tested according to Baumann and Baumann [15], using supplementation with 1 % (v/v) vitamin solution [16] and 2 % (v/ v) Hutner’ s mineral salts [17]. Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (µg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin G (20 IU), polymyxin B (100 IU), streptomycin (50) and tetracycline (30). Enzyme activities were determined, after incubation for 10 h at 20 Ǫ C, by using the API ZYM system (bioḾerieux); the strip was inoculated with cells suspended in artificial seawater from which CaCl 2 was excluded to avoid the formation of precipitates.Cell mass of strain RA2-7 T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in MB at 20 Ǫ C. Chromosomal DNA was extracted and purified as described previously [18], with the exception that RNase T 1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR as described previously [19] using two universal primers, 9F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1512R (5′- ACGGTTACCTTGTTACGACTT-3′). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. [20]. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. [21] using 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 mean of the remaining three values was quoted as DNA–DNA relatedness value. The DNA of strain RA2-7 T was used as a labelled DNA probe.Isoprenoid quinones were extracted according to the method of Komagata and Suzuki [22] and analysed using reversed-phase HPLC and a YMC ODS-A (250× 4.6 mm) column. The isoprenoid quinones were eluted by a mixture of methanol/isopropanol (2: 1, v/v) using a flow rate of 1 ml min ‒1 at room temperature, and were detected by UV absorbance at 275 nm. For cellular fatty acids analysis, cell masses of strain RA2-7 T were harvested from MA plates after cultivation for 3, 5 and 7 days at 20 Ǫ C. Fatty acids were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.2B). The fatty acids were analysed by GC (6890; Hewlett Packard) and identified using the TSBA6 database of the Microbial Identification System [23]. Cell masses of C. aestuarii SMK-10 T, C. polaris JCM 13952 T and C. chukchiensis LMG 25329 T for analysis of polar lipids were obtained from cultures grown for 3 days in MB at 25 Ǫ C. Polar lipids were extracted according to the procedures described by Minnikin et al. [24], and separated by two-dimensional TLC using chloroform/methanol/water (65: 25: 3.8, by vol.) for the first dimension and chloroform/ methanol/acetic acid/water (40: 7.5: 6: 1.8, by vol.) for the second dimension as described by Embley and Wait [25]. Individual polar lipids were identified by spraying the plates with 10 % ethanolic molybdophosphoric acid and a- naphthol reagents [24] and with the ninhydrin spray, molybdenum blue spray and Dragendorff’ s reagents (Sigma). The DNA G+C content was determined by the method of Tamaoka and Komagata [26] with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC equipped with a YMC ODS-A (250× 4.6 mm) column. The nucleotides were eluted by a mixture of 0.55 M NH 4 H 2 PO 4 (pH 4.0) and acetonitrile (40: 1, v/v), using a flow rate of 1 ml min ‒1 at room temperature, and were detected by UV absorbance at 270 nm. The mean of three replications was quoted as the DNA G+C content value. DNA from Escherichia coli (Sigma) was used as a standard and for calibration.Morphological, cultural, physiological and biochemical characteristics of strain RA2-7 T are given in the species description and in Table 1 or Fig. S1 (available in the online Supplementary Material). The almost-complete 16S rRNA gene sequence of strain RA2-7 T comprising 1463 nucleotides, representing approximately 95 % of the E. coli 16S rRNA gene sequence, was determined in this study. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain RA2-7 T fell within the clade comprising the type strains of species of the genus Colwellia, clustering coherently with the type strains of C. sediminilitoris, C. aestuarii, C. polaris and C. chukchiensis (Fig. 1). The clustering of strain RA2-7 T and the type strains of these four species of the genus Colwellia was also found in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain RA2-7 T exhibited 16S rRNA gene sequence similarity values of 98.3, 98.0, 97.5 and 96.3 % to C. sediminilitoris YSM-23 T, C. aestuarii SMK-10 T, C. polaris 537 T and C. chukchiensis BCw111 T, respectively, and of 94.5–96.5 % to the type strains of the other species of the genus Colwellia.The predominant isoprenoid quinone detected in strain RA2-7 T was ubiquinone-8 (Q-8), which is the same as that shown in the genus Colwellia [3, 7 – 10, 27 – 29]. In Table 2, the cellular fatty acid profiles of strain RA2-7 T are compared with those of the type strains of C. sediminilitoris, C. aestuarii, C. polaris and C. chukchiensis. The fatty acid profiles of strain RA2-7 T from the three different growth phases were found to be similar (Table 2). The major fatty acids (&gt;10 % of the total fatty acids in all growth phases) detected in strain RA2-7 T were summed feature 3 (C 16: 1 Ɯ7c and/or C 16: 1 Ɯ6c) and C 16: 0 (Table 2). The fatty acid profile of strain RA2-7 T was similar to those of the type strains of the four phylogenetically closely related species of the genus Colwellia, although there were differences in the proportions of some fatty acids (Table 2). The major polar lipids detected in strain RA2-7 T were phosphatidylglycerol and phosphatidylethanolamine; minor amounts of one unidentified aminophospholipid, six unidentified phospholipids and two unidentified lipids were also present (Fig. S2). The major polar lipid profile of strain RA2-7 T was similar to those of the type strains of C. sediminilitoris, C. aestuarii, C. polaris and C. chukchiensis, the closest phylogenetic relatives, in that phosphatidylglycerol and phosphatidylethanolamine are major components (Fig. S2; [3]). The DNA G+C content of strain RA2-7 T was 39.0±0.04 mol%, a value in the range reported for species of the genus Colwellia (Table 1; [1, 4]).Table 1. Differential characteristics of strain RA2-7T and the type strains of four phylogenetically closely related species of the genus Colwellia Strains: 1, RA2-7T (data from this study); 2, C. sediminilitoris YSM-23T (data from Park et al. [3]); 3, C. aestuarii SMK-10T (Jung et al. [6]); 4, C. polaris JCM 13952T (Kim et al. [9]); 5, C. chukchiensis LMG 25329T (Kim et al. [9]). +, Positive; –, negative;, weakly positive. All strains are positive for motil- W ity*; activity of* catalase and oxidase; hydrolysis of† casein and Tween 80; activity of alkaline phosphatase and leucine arylamidase; and susceptibility to cephalothin, chloramphenicol, gentamicin, oleandomycin, polymyxin B and streptomycin. All strains are negative for Gram staining; growth at 37 Ǫ C; hydrolysis of xanthine†; utilization of -fructose, -mannose, benzoate, formate and succinate; activity of lipase (C14), cystine arylamidase, tryp- DDsin, a- chymotrypsin, a- galactosidase, b- galactosidase, b- glucuronidase, a- glucosidase, N -acetyl-b- glucosaminidase, a- mannosidase and a- fucosidase; and susceptibility to lincomycin.Characteristic 1 2 3 4 5 Optimal growth temperature (Ǫ C)* 20 30 25–30 20–21 a 23–25 b Nitrate reduction† ‒ + + ‒ + Hydrolysis of:† Aesculin ‒ ‒ ‒ + + Gelatin ‒ + ‒ + + Hypoxanthine ‒ + ‒ + ‒ Starch + + ‒ + ‒ L- Tyrosine ‒ + ‒ + + Urea ‒ ‒ ‒ ‒ + Utilization of: L- Arabinose, cellobiose, trehalose, D- xylose, L- malate, salicin ‒ ‒ + ‒ ‒ D- Galactose, D- glucose ‒ ‒ + + + Maltose ‒ ‒ + + ‒ Sucrose ‒ ‒ ‒ + ‒ Acetate, L- glutamate ‒ ‒ ‒ + + Citrate ‒ ‒ ‒ ‒ + Pyruvate + ‒ ‒ + + Enzyme activity (API ZYM) Esterase (C4) + + + ‒ ‒ Esterase lipase (C8) + + + W ‒ Valine arylamidase ‒ ‒ ‒ W ‒ Acid phosphatase + ‒ + + ‒ Naphthol-AS-BI-phosphohydrolase W ‒ + + W b- Glucosidase ‒ ‒ + ‒ ‒ Susceptibility to: Ampicillin + + ‒ + ‒ Carbenicillin + + + + ‒ Kanamycin ‒ + + ‒ + Neomycin ‒ + + + + Novobiocin + + + ‒ + Penicillin G + ‒ + + ‒ Tetracycline ‒ + ‒ ‒ + DNA G+C content (mol%)* 39.0±0.04 43.8±0.08 39.3 38.9 a 41.3 b*Data of reference strains taken from: a, Zhang et al. [27]; b, Yu et al. [8]. †Data of C. polaris JCM 13952T and C. chukchiensis LMG 25329T obtained from this study.Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Colwellia mytili sp. nov. RA2- 7 T, the type strains of other species of the genus Colwellia and representatives of some other related taxa. Bootstrap values (expressed as percentages of 1000 replications)&gt;50 % are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Pseudomonas aeruginosa DSM 50071 T (GenBank accession number X06684) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.The results obtained from the phylogenetic and chemotaxonomic analyses are sufficient to support that strain RA2-7 T is a member of the genus Colwellia (Figs 1, S2 and Table 2). Strain RA2-7 T exhibited DNA–DNA relatedness values of 19, 14 and 17 % to C. sediminilitoris YSM-23 T, C. aestuarii SMK-10 T and C. polaris JCM 13952 T, respectively. Strain RA2-7 T was distinguished from the type strains of C. sediminilitoris, C. aestuarii, C. polaris and C. chukchiensis by differences in several phenotypic characteristics, including nitrate reduction, utilization of some substrates, activity of some enzymes and susceptibility to some antibiotics (Table 1). These differences, together with the phylogenetic and genetic distinctiveness of strain RA2-7 T, suggest that the novel strain is separated from other species of the genus Colwellia [30, 31]. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain RA2-7 T is considered to represent a novel species of the genus Colwellia, for which the name Colwellia mytili sp. nov. is proposed.