International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1520–1525
DOI 10.1099/ijs.0.060467-0
Winogradskyella wandonensis sp. nov., isolated from a tidal flat Sooyeon Park,1 Ji-Min Park,1 Sung-Min Won,1 Kyung Sook Bae2 and Jung-Hoon Yoon1 Correspondence Jung-Hoon Yoon [email protected]
1
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
2
Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yuseong, Daejeon, Republic of Korea
A Gram-stain-negative, non-flagellated, non-gliding, aerobic, rod-shaped bacterium, designated WD-2-2T, was isolated from a tidal flat of Wando, an island of South Korea, and subjected to a polyphasic taxonomic analysis. Strain WD-2-2T grew optimally at 30 6C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain WD-2-2T belonged to the genus Winogradskyella, clustering coherently with the type strain of Winogradskyella litorisediminis. Strain WD-2-2T exhibited 16S rRNA gene sequence similarity of 97.4 % to W. litorisediminis DPS-8T and 94.5–96.6 % to the type strains of the other species of the genus Winogradskyella. Strain WD-2-2T contained MK-6 as the predominant menaquinone and iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0 as the major fatty acids. The major polar lipids detected in strain WD-2-2T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid. The DNA G+C content was 36.4 mol%, and DNA–DNA relatedness with W. litorisediminis DPS-8T was 13 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain WD-22T is separate from recognized species of the genus Winogradskyella. On the basis of the data presented, strain WD-2-2T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella wandonensis sp. nov. is proposed. The type strain is WD-2-2T (5KCTC 32579T5CECT 8445T).
The genus Winogradskyella, a member of family Flavobacteriaceae (phylum Bacteroidetes), was proposed by Nedashkovskaya et al. (2005) with the descriptions of three novel species, Winogradskyella thalassocola (the type species of the genus), W. epiphytica and W. eximia. At the time of writing, the genus comprised 17 species with validly published names (http://www.bacterio.net/winogradskyella.html; Euze´by, 1997). All known members of the genus Winogradskyella have been isolated from marine environments or marine organisms (Lau et al., 2005; Nedashkovskaya et al., 2005, 2009, 2012; Pinhassi et al., 2009; Romanenko et al., 2009; Ivanova et al., 2010; Kim & Nedashkovskaya, 2010; Yoon et al., 2011; Lee et al., 2012; Yoon & Lee, 2012; Begum et al., 2013; Lee et al., 2013). In this study, we describe a Winogradskyella-like bacterial strain, designated WD-2-2T, collected from a tidal flat sediment. The aim of the present work was to determine the exact taxonomic position of strain WD-2-2T by polyphasic taxonomic characterization which included determination of chemotaxonomic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain WD-2-2T is KF768343. A supplementary figure is available with the online version of this paper.
1520
and other phenotypic properties, detailed phylogenetic investigations based on 16S rRNA gene sequences and DNA–DNA hybridization. A sample of tidal flat sediment was collected from Wando, an island of South Korea, on the South Sea, and used as the source for isolation of bacterial strains. Strain WD-2-2T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson) and cultivated routinely at 30 uC on MA. Winogradskyella litorisediminis DPS-8T, which was used as a reference strain for fatty acid analysis and DNA–DNA hybridization, was obtained in our previous study (Kang et al., 2013). Cell morphology was examined by light microscopy (BX51; Olympus) and transmission electron microscopy (JEM1010; JEOL). The latter technique was also used to assess the presence of flagella on cells from an exponentially growing MA culture. For this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and grids were examined after being air-dried. Gliding motility was investigated as described by Bowman (2000). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions 060467 G 2014 IUMS Printed in Great Britain
Winogradskyella wandonensis sp. nov.
was determined after incubation for 10 days in an anaerobic jar (MGC) with AnaeroPack (MGC) on MA and on MA with potassium nitrate (0.1 %, w/v); the jar was kept overnight at 4 uC to create anoxic conditions before incubation at 30 uC. Growth on MA at 4, 10, 15, 20, 25, 30, 37, 40 and 45 uC was assessed to determine the optimal temperature and temperature range for growth. The pH range for growth was determined in marine broth 2216 (MB; Becton Dickinson) adjusted to pH 4.5–9.5 (in increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH was verified after autoclaving. Growth at various concentrations of NaCl (0, 0.5 and 1.0– 8.0 %, in increments of 1.0 %) was investigated by adding appropriate amounts of NaCl to MB prepared according to the formula of the BD medium except that NaCl was omitted. Requirement for Mg2+ ions was investigated by using MB prepared according to the formula of the BD medium except that MgCl2 and MgSO4 were omitted. Catalase and oxidase activities were determined as described by La´nyi (1987). Hydrolysis of casein, starch, hypoxanthine, L-tyrosine and xanthine was investigated on MA using the substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously (La´nyi, 1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea was investigated by using nutrient gelatin and urea agar base media (Becton Dickinson), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l21 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). The presence of flexirubin-type pigments was investigated as described previously (Reichenbach, 1992; Bernardet et al., 2002). Acid production from carbohydrates was tested as described by Leifson (1963). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (mg 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 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined by using the API ZYM system (bioMe´rieux) after incubation for 8 h at 30 uC. Cell biomass of strain WD-2-2T for DNA extraction and for analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 30 uC. Chromosomal DNA was extracted and purified as described previously (Yoon et al., 1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination by RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers (59-GAGTTTGATCCTGGCTCAG-39 and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA http://ijs.sgmjournals.org
hybridization was performed fluorometrically by the method of Ezaki et al. (1989) 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 means of the remaining three values are quoted as DNA–DNA relatedness values. DNA from strain WD-2-2T and W. litorisediminis DPS-8T was used individually as the labelled DNA probe for reciprocal hybridization. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (25064.6 mm) column. Isoprenoid quinones were eluted with methanol/ 2-propanol (2 : 1, v/v) at a flow rate of 1 ml min21 at room temperature and detected by monitoring the A270. For cellular fatty acid analysis, cell mass of strain WD-2-2T and W. litorisediminis DPS-8T was harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell mass was standardized by observing the growth development of colonies on agar plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified using the TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) 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 Minnikin et al. (1977). Individual polar lipids were identified by spraying the plates with 10 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s reagent (Sigma). 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 equipped with a YMC ODS-A (25064.6 mm) column. Nucleotides were eluted with a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v) at a flow rate of 1 ml min21 at room temperature and detected by monitoring the A270. Morphological, cultural, physiological and biochemical characteristics of strain WD-2-2T are given in the species description and in Table 1. The almost-complete 16S rRNA gene sequence of strain WD-2-2T determined in this study comprised 1448 nt, representing approximately 95 % of the 16S rRNA gene sequence of Escherichia coli. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain WD-2-2T fell within the clade comprising species of the genus Winogradskyella, particularly clustering coherently with the type strain of W. 1521
S. Park and others
Table 1. Differential characteristics of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola
litorisediminis DPS-8T and 94.5–96.6 % to the type strains of the other species of the genus Winogradskyella.
Strains: 1, WD-2-2T; 2, W. litorisediminis DPS-8T (data from Kang et al., 2013); 3, W. thalassocola KCTC 12221T (unless indicated otherwise, data from Yoon & Lee, 2012). All three strains are positive for the following: activities of catalase and oxidase; hydrolysis of casein, gelatin and Tweens 20, 40, 60 and 80; acid production from Dglucose, maltose and D-mannose; susceptibility to carbenicillin, chloramphenicol, lincomycin and oleandomycin; and activities of alkaline phosphatase, leucine arylamidase and naphthol-AS-BIphosphohydrolase. Activities of esterase (C4), esterase lipase (C8) and acid phosphatase are positive for all three strains, but only weakly positive for W. thalassocola KCTC 12221T. All three strains are negative for the following: anaerobic growth; Gram-staining; nitrate reduction; H2S production; production of flexirubin-type pigments; hydrolysis of urea, hypoxanthine and xanthine; acid production from L-arabinose, D-fructose, D-galactose, lactose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, trehalose, D-xylose, myo-inositol, Dmannitol and D-sorbitol; susceptibility to gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activities of lipase (C14), a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase.
The predominant isoprenoid quinone detected in strain WD-2-2T was menaquinone-6 (MK-6), in line with other members of the genus Winogradskyella (Nedashkovskaya et al., 2005, 2012; Begum et al., 2013) and all other members of the family Flavobacteriaceae (Bernardet, 2011). The cellular fatty acid profiles of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola are compared in Table 2. The major fatty acids (.10 % of the total fatty acids) detected in strain WD-2-2T were iso-C15 : 1 G (21.8 %), iso-C17 : 0 3-OH (21.5 %) and iso-C15 : 0 (15.9 %). The fatty acid profiles of the three strains were similar, although there were differences in the proportions of some fatty acids and in the presence or absence of some fatty acids (Table 2). The major polar lipids detected in strain WD-2-2T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid (Fig. S1, available in the online Supplementary Material). The polar lipid profile of strain WD-2-2T was similar to those of W. litorisediminis DPS-8T and W. thalassocola KCTC 12221T in that the only identified phospholipid is phosphatidylethanolamine, and one unidentified lipid is a major polar lipid, although they were distinguishable from each other by the presence or absence of several unidentified lipids (Lee et al., 2012; Kang et al., 2013). The DNA G+C content of strain WD-2-2T was 36.4 mol%, a value slightly higher than that of W. litorisediminis DPS-8T but in the range reported for members of the genus Winogradskyella (Table 1; Kang et al., 2013; Begum et al., 2013). The results obtained from the phylogenetic and chemotaxonomic analyses are sufficient to determine the taxonomic position of strain WD2-2T as a member of the genus Winogradskyella.
Characteristic Growth at: 4 and 10 uC 37 and 40 uC Hydrolysis of: Aesculin Starch L-Tyrosine Acid production from: Cellobiose Sucrose Susceptibility to: Ampicillin Cephalothin Novobiocin Penicillin G Tetracycline Enzyme activity (API ZYM) Valine arylamidase Cystine arylamidase Trypsin a-Chymotrypsin DNA G+C content (mol%)
1
2
3
2 +
+ 2
+* 2*
2 2 +
+ + 2
+ 2 +
2 2
2 +
+ +
+ + + + 2
2 2 + 2 +
2 + 2 2 2
+ + + + 36.4
+ + + 2 34.7
2 2 2 2 34.6*
*Data taken from Nedashkovskaya et al. (2005).
Mean DNA–DNA relatedness between strain WD-2-2T and W. litorisediminis DPS-8T was 13 %. Strain WD-2-2T was distinguished from the type strains of W. litorisediminis and W. thalassocola by differences in several phenotypic characteristics, including the temperature for growth, hydrolysis of some substrates, acid production from some substrates, susceptibility to some antibiotics and activities of some enzymes (Table 1). These differences, in combination with the phylogenetic and genetic distinctiveness of strain WD-2-2T, suggest that the novel strain is separate from other species of the genus Winogradskyella (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, strain WD-2-2T is therefore considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella wandonensis sp. nov. is proposed. Description of Winogradskyella wandonensis sp. nov.
litorisediminis with a bootstrap resampling value of 96.7 % (Fig. 1). The relationship between strain WD-2-2T and W. litorisediminis DPS-8T was maintained in trees reconstructed using the maximum-likelihood and maximumparsimony algorithms (Fig. 1). Strain WD-2-2T exhibited 16S rRNA gene sequence similarity of 97.4 % to W. 1522
Winogradskyella wandonensis (wan.do.nen9sis. N.L. fem. adj. wandonensis pertaining to Wando, an island of South Korea, where the type strain was isolated). Cells are Gram-stain-negative, non-flagellated, non-gliding and rod-shaped, approximately 0.2–0.5 mm wide and International Journal of Systematic and Evolutionary Microbiology 64
Winogradskyella wandonensis sp. nov.
Winogradskyella undariae WS-MY5T (KC261665)
58.6
0.01
Winogradskyella pacifica KMM 6019T (GQ181061) Winogradskyella psychrotolerans RS-3T (FN377721) Winogradskyella thalassocola KMM 3907T (AY521223) Winogradskyella rapida SCB 36T (U64013) 100 Winogradskyella arenosi R 60T (AB438962)
72.7
Winogradskyella damuponensis F081-2T (HQ336488) Winogradskyella eximia KMM 3944T (AY521225) Winogradskyella multivorans T-Y1T (JQ354979) Winogradskyella lutea A73T (FJ919968)
59.7
Winogradskyella epiphytica KMM 3906T (AY521224) 98.7 79.4
69.2
Winogradskyella pulchriflava EM106T (JN896598) Winogradskyella echinorum KMM 6211T (EU727254) Winogradskyella ulvae KMM 6390T (HQ456127)
97.4 87.5
Winogradskyella aquimaris DPG-24T (HM368527) Winogradskyella poriferorum UST030701-295T (AY848823)
Winogradskyella exilis 022-2-26T (FJ595484) Winogradskyella wandonensis WD-2-2T (KF768343)
96.7
Winogradskyella litorisediminis DPS-8T (JQ432561) Marinivirga aestuarii KYW371T (HQ405792)
84.1 76.7
Pontirhabdus pectinovorans JC2675T (HM475134) Postechiella marina M091T (HQ336487) Lacinutrix copepodicola DJ3T (AY694001) Lacinutrix himadriensis E4-9aT (FN377744)
100
Gaetbulibacter saemankumensis SMK-12T (AY883937)
62.9 58.5
Gaetbulibacter marinus IMCC1914T (EF108219) Meridianimaribacter flavus NH57NT (FJ360684) 100
Mariniflexile gromovii KMM 6038T (DQ312294) Mariniflexile fucanivorans SW5T (AJ628046) Flaviramulus basaltis H35T (DQ361033) Psychroserpens burtonensis ACAM 188T (U62913)
100
Psychroserpens damuponensis F051-1T (HQ336490) Capnocytophaga ochracea ATCC 27872T (U41350)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Winogradskyella wandonensis sp. nov. WD-2-2T and representatives of other species of the genus Winogradskyella and of some other related taxa. Only bootstrap values greater than 50 % (expressed as percentages of 1000 replications) are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in trees reconstructed with the maximum-likelihood and maximum-parsimony algorithms. Capnocytophaga ochracea ATCC 27872T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
0.7–10.0 mm long. Colonies on MA are circular, slightly convex, smooth, glistening, strong orange–yellow and 1.0– 1.5 mm in diameter after incubation for 3 days at 30 uC. The optimal temperature for growth is 30 uC; growth occurs at 15 and 40 uC but not at 10 or 45 uC. Optimal growth occurs at pH 7.0–8.0; growth occurs at pH 6.0 but not at pH 5.5. Optimal growth occurs in the presence of approximately 2.0 % (w/v) NaCl; growth occurs in the presence of 0.5–5.0 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Flexirubin-type pigments are not produced. H2S is not produced. Casein, gelatin, Tweens 20, 40, 60 and 80 and http://ijs.sgmjournals.org
L-tyrosine
are hydrolysed, but aesculin, hypoxanthine, starch, urea and xanthine are not. Acid is produced from D-glucose, maltose and D-mannose, but not from Larabinose, cellobiose, D-fructose, D-galactose, lactose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, sucrose, trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. In assays with the API ZYM system, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase and naphthol-AS-BIphosphohydrolase are present, but activities of lipase (C14), a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, 1523
S. Park and others
Table 2. Cellular fatty acid compositions of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola T
T
Strains: 1, WD-2-2 ; 2, W. litorisediminis DPS-8 ; 3, W. thalassocola KCTC 12221T (data from Park & Yoon, 2013). Data were obtained from this study unless indicated. Values are percentages of total fatty acids; fatty acids that represented ,0.5 % in all strains were omitted. TR, Trace (,0.5 %); 2, not detected. Fatty acid Straight-chain C14 : 0 C16 : 0 C18 : 0 Branched iso-C14 : 0 iso-C15 : 0 iso-C15 : 1 G* anteiso-C15 : 0 anteiso-C15 : 1 A* iso-C16 : 0 iso-C16 : 1 G* iso-C16 : 1 H* Hydroxy C15 : 0 2-OH C15 : 0 3-OH C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH iso-C13 : 0 3-OH iso-C14 : 0 3-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Unsaturated C15 : 1v6c C17 : 1v6c C18 : 1v5c iso-C17 : 1v9c Summed features Summed feature 3D
1
2
3
Acknowledgements This work was supported by the Program for Collection, Management and Utilization of Biological Resources from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (grant NRF2013M3A9A5075953).
References Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel’s Manual for
the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. 0.9 2
2 0.9 2
1.1 1.5 0.7
0.8 15.9 21.8 1.3 1.6 1.4 1.9 2
0.8 17.2 25.0 2.2 1.7 2.0 0.8 2
1.3 17.7 15.0 11.4 2.6 1.2 2 2.2
1.2 0.7 1.8 0.9 0.8
0.9 0.7 1.1 1.1 0.9 0.8
1.4 1.5 0.8 2.6 2
TR
TR
TR
Begum, Z., Srinivas, T. N. R., Manasa, P., Sailaja, B., Sunil, B., Prasad, S. & Shivaji, S. (2013). Winogradskyella psychrotolerans sp.
nov., a marine bacterium of the family Flavobacteriaceae isolated from Arctic sediment. Int J Syst Evol Microbiol 63, 1646–1652. Bernardet, J.-F. (2011). Family I. Flavobacteriaceae Reichenbach 1992. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 4, pp. 106–111. Edited by N. R. Krieg, W. Ludwig, W. B. Whitman, B. P. Hedlund, B. J. Paster, J. T. Staley, N. Ward, D. Brown & A. Parte. New York: Springer. Bernardet, J.-F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002).
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861– 1868.
0.6 7.8 8.1 21.5
6.9 5.7 19.9
2 10.8 10.4 8.4
2 0.7 2 2
2 2 2 2
2.1 0.7 0.9 2.1
9.8
10.8
3.0
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
*Double bond position indicated by a capital letter is unknown. DSummed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Ivanova, E. P., Christen, R., Gorshkova, N. M., Zhukova, N. V., Kurilenko, V. V., Crawford, R. J. & Mikhailov, V. V. (2010).
a-mannosidase and a-fucosidase are absent. Susceptible to
Winogradskyella exilis sp. nov., isolated from the starfish Stellaster equestris, and emended description of the genus Winogradskyella. Int J Syst Evol Microbiol 60, 1577–1580.
TR
ampicillin, carbenicillin, cephalothin, chloramphenicol, lincomycin, novobiocin, oleandomycin and penicillin G, but not to gentamicin, kanamycin, neomycin, polymyxin B, streptomycin or tetracycline. The predominant menaquinone is MK-6. The major fatty acids (.10 % of the total fatty acids) are iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0. The polar lipids are phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid. The type strain, WD-2-2T (5KCTC 32579T5CECT 8445T), was isolated from a tidal flat at Wando in the South Sea of South Korea. The DNA G+C content of the type strain is 36.4 mol%. 1524
Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Euze´by, J. P. (1997). List of Bacterial Names with Standing in
Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 47, 590–592. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
Kang, C.-H., Lee, S.-Y. & Yoon, J.-H. (2013). Winogradskyella
litorisediminis sp. nov., isolated from coastal sediment. Int J Syst Evol Microbiol 63, 1793–1799. Kim, S. B. & Nedashkovskaya, O. I. (2010). Winogradskyella pacifica
sp. nov., a marine bacterium of the family Flavobacteriaceae. Int J Syst Evol Microbiol 60, 1948–1951. Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207. La´nyi, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67. Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Dobretsov, S., Lau, K. W. K., Wu, M., Wong, P.-K., Pawlik, J. R. & Qian, P.-Y. (2005).
Winogradskyella poriferorum sp. nov., a novel member of the family International Journal of Systematic and Evolutionary Microbiology 64
Winogradskyella wandonensis sp. nov. Flavobacteriaceae isolated from a sponge in the Bahamas. Int J Syst Evol Microbiol 55, 1589–1592.
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
Lee, S.-Y., Park, S., Oh, T.-K. & Yoon, J.-H. (2012). Winogradskyella
2nd edn, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
aquimaris sp. nov., isolated from seawater. Int J Syst Evol Microbiol 62, 1814–1818.
Romanenko, L. A., Tanaka, N., Frolova, G. M. & Mikhailov, V. V. (2009). Winogradskyella arenosi sp. nov., a member of the family
Lee, D.-H., Cho, S. J., Kim, S. M. & Lee, S. B. (2013). Winogradskyella
Flavobacteriaceae isolated from marine sediments from the Sea of Japan. Int J Syst Evol Microbiol 59, 1443–1446.
damuponensis sp. nov., isolated from seawater. Int J Syst Evol Microbiol 63, 321–326.
Sasser, M. (1990). Identification of bacteria by gas chromatography of
Leifson, E. (1963). Determination of carbohydrate metabolism of
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI, Inc.
marine bacteria. J Bacteriol 85, 1183–1184.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977).
DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846– 849.
Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol 27, 104–117. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Snauwaert, C., Vancanneyt, M., Swings, J., Kim, K.-O., Lysenko, A. M., Rohde, M. & other authors (2005). Winogradskyella thalassocola gen. nov., sp.
nov., Winogradskyella epiphytica sp. nov. and Winogradskyella eximia sp. nov., marine bacteria of the family Flavobacteriaceae. Int J Syst Evol Microbiol 55, 49–55. Nedashkovskaya, O. I., Vancanneyt, M., Kim, S. B. & Zhukova, N. V. (2009). Winogradskyella echinorum sp. nov., a marine bacterium of the
family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius. Int J Syst Evol Microbiol 59, 1465–1468. Nedashkovskaya, O. I., Kukhlevskiy, A. D. & Zhukova, N. V. (2012).
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. Yoon, J.-H. & Lee, S.-Y. (2012). Winogradskyella multivorans sp. nov.,
a polysaccharide-degrading bacterium isolated from seawater of an oyster farm. Antonie van Leeuwenhoek 102, 231–238. Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of
Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Winogradskyella ulvae sp. nov., an epiphyte of a Pacific seaweed, and emended descriptions of the genus Winogradskyella and Winogradskyella thalassocola, Winogradskyella echinorum, Winogradskyella exilis and Winogradskyella eximia. Int J Syst Evol Microbiol 62, 1450–1456.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
Park, S. & Yoon, J.-H. (2013). Winogradskyella undariae sp. nov., a
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
member of the family Flavobacteriaceae isolated from a brown algae reservoir. Antonie van Leeuwenhoek 104, 619–626. Pinhassi, J., Nedashkovskaya, O. I., Hagstro¨m, A. & Vancanneyt, M. (2009). Winogradskyella rapida sp. nov., isolated from protein-
enriched seawater. Int J Syst Evol Microbiol 59, 2180–2184.
http://ijs.sgmjournals.org
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194. Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp.
Yoon, B.-J., Byun, H.-D., Kim, J.-Y., Lee, D.-H., Kahng, H.-Y. & Oh, D.-C. (2011). Winogradskyella lutea sp. nov., isolated from seawater,
and emended description of the genus Winogradskyella. Int J Syst Evol Microbiol 61, 1539–1543.
1525
DOI 10.1099/ijs.0.060467-0
Winogradskyella wandonensis sp. nov., isolated from a tidal flat Sooyeon Park,1 Ji-Min Park,1 Sung-Min Won,1 Kyung Sook Bae2 and Jung-Hoon Yoon1 Correspondence Jung-Hoon Yoon [email protected]
1
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
2
Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yuseong, Daejeon, Republic of Korea
A Gram-stain-negative, non-flagellated, non-gliding, aerobic, rod-shaped bacterium, designated WD-2-2T, was isolated from a tidal flat of Wando, an island of South Korea, and subjected to a polyphasic taxonomic analysis. Strain WD-2-2T grew optimally at 30 6C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain WD-2-2T belonged to the genus Winogradskyella, clustering coherently with the type strain of Winogradskyella litorisediminis. Strain WD-2-2T exhibited 16S rRNA gene sequence similarity of 97.4 % to W. litorisediminis DPS-8T and 94.5–96.6 % to the type strains of the other species of the genus Winogradskyella. Strain WD-2-2T contained MK-6 as the predominant menaquinone and iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0 as the major fatty acids. The major polar lipids detected in strain WD-2-2T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid. The DNA G+C content was 36.4 mol%, and DNA–DNA relatedness with W. litorisediminis DPS-8T was 13 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain WD-22T is separate from recognized species of the genus Winogradskyella. On the basis of the data presented, strain WD-2-2T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella wandonensis sp. nov. is proposed. The type strain is WD-2-2T (5KCTC 32579T5CECT 8445T).
The genus Winogradskyella, a member of family Flavobacteriaceae (phylum Bacteroidetes), was proposed by Nedashkovskaya et al. (2005) with the descriptions of three novel species, Winogradskyella thalassocola (the type species of the genus), W. epiphytica and W. eximia. At the time of writing, the genus comprised 17 species with validly published names (http://www.bacterio.net/winogradskyella.html; Euze´by, 1997). All known members of the genus Winogradskyella have been isolated from marine environments or marine organisms (Lau et al., 2005; Nedashkovskaya et al., 2005, 2009, 2012; Pinhassi et al., 2009; Romanenko et al., 2009; Ivanova et al., 2010; Kim & Nedashkovskaya, 2010; Yoon et al., 2011; Lee et al., 2012; Yoon & Lee, 2012; Begum et al., 2013; Lee et al., 2013). In this study, we describe a Winogradskyella-like bacterial strain, designated WD-2-2T, collected from a tidal flat sediment. The aim of the present work was to determine the exact taxonomic position of strain WD-2-2T by polyphasic taxonomic characterization which included determination of chemotaxonomic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain WD-2-2T is KF768343. A supplementary figure is available with the online version of this paper.
1520
and other phenotypic properties, detailed phylogenetic investigations based on 16S rRNA gene sequences and DNA–DNA hybridization. A sample of tidal flat sediment was collected from Wando, an island of South Korea, on the South Sea, and used as the source for isolation of bacterial strains. Strain WD-2-2T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson) and cultivated routinely at 30 uC on MA. Winogradskyella litorisediminis DPS-8T, which was used as a reference strain for fatty acid analysis and DNA–DNA hybridization, was obtained in our previous study (Kang et al., 2013). Cell morphology was examined by light microscopy (BX51; Olympus) and transmission electron microscopy (JEM1010; JEOL). The latter technique was also used to assess the presence of flagella on cells from an exponentially growing MA culture. For this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and grids were examined after being air-dried. Gliding motility was investigated as described by Bowman (2000). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions 060467 G 2014 IUMS Printed in Great Britain
Winogradskyella wandonensis sp. nov.
was determined after incubation for 10 days in an anaerobic jar (MGC) with AnaeroPack (MGC) on MA and on MA with potassium nitrate (0.1 %, w/v); the jar was kept overnight at 4 uC to create anoxic conditions before incubation at 30 uC. Growth on MA at 4, 10, 15, 20, 25, 30, 37, 40 and 45 uC was assessed to determine the optimal temperature and temperature range for growth. The pH range for growth was determined in marine broth 2216 (MB; Becton Dickinson) adjusted to pH 4.5–9.5 (in increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH was verified after autoclaving. Growth at various concentrations of NaCl (0, 0.5 and 1.0– 8.0 %, in increments of 1.0 %) was investigated by adding appropriate amounts of NaCl to MB prepared according to the formula of the BD medium except that NaCl was omitted. Requirement for Mg2+ ions was investigated by using MB prepared according to the formula of the BD medium except that MgCl2 and MgSO4 were omitted. Catalase and oxidase activities were determined as described by La´nyi (1987). Hydrolysis of casein, starch, hypoxanthine, L-tyrosine and xanthine was investigated on MA using the substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously (La´nyi, 1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea was investigated by using nutrient gelatin and urea agar base media (Becton Dickinson), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l21 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). The presence of flexirubin-type pigments was investigated as described previously (Reichenbach, 1992; Bernardet et al., 2002). Acid production from carbohydrates was tested as described by Leifson (1963). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (mg 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 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined by using the API ZYM system (bioMe´rieux) after incubation for 8 h at 30 uC. Cell biomass of strain WD-2-2T for DNA extraction and for analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 30 uC. Chromosomal DNA was extracted and purified as described previously (Yoon et al., 1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination by RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers (59-GAGTTTGATCCTGGCTCAG-39 and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA http://ijs.sgmjournals.org
hybridization was performed fluorometrically by the method of Ezaki et al. (1989) 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 means of the remaining three values are quoted as DNA–DNA relatedness values. DNA from strain WD-2-2T and W. litorisediminis DPS-8T was used individually as the labelled DNA probe for reciprocal hybridization. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (25064.6 mm) column. Isoprenoid quinones were eluted with methanol/ 2-propanol (2 : 1, v/v) at a flow rate of 1 ml min21 at room temperature and detected by monitoring the A270. For cellular fatty acid analysis, cell mass of strain WD-2-2T and W. litorisediminis DPS-8T was harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell mass was standardized by observing the growth development of colonies on agar plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified using the TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) 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 Minnikin et al. (1977). Individual polar lipids were identified by spraying the plates with 10 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s reagent (Sigma). 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 equipped with a YMC ODS-A (25064.6 mm) column. Nucleotides were eluted with a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v) at a flow rate of 1 ml min21 at room temperature and detected by monitoring the A270. Morphological, cultural, physiological and biochemical characteristics of strain WD-2-2T are given in the species description and in Table 1. The almost-complete 16S rRNA gene sequence of strain WD-2-2T determined in this study comprised 1448 nt, representing approximately 95 % of the 16S rRNA gene sequence of Escherichia coli. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain WD-2-2T fell within the clade comprising species of the genus Winogradskyella, particularly clustering coherently with the type strain of W. 1521
S. Park and others
Table 1. Differential characteristics of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola
litorisediminis DPS-8T and 94.5–96.6 % to the type strains of the other species of the genus Winogradskyella.
Strains: 1, WD-2-2T; 2, W. litorisediminis DPS-8T (data from Kang et al., 2013); 3, W. thalassocola KCTC 12221T (unless indicated otherwise, data from Yoon & Lee, 2012). All three strains are positive for the following: activities of catalase and oxidase; hydrolysis of casein, gelatin and Tweens 20, 40, 60 and 80; acid production from Dglucose, maltose and D-mannose; susceptibility to carbenicillin, chloramphenicol, lincomycin and oleandomycin; and activities of alkaline phosphatase, leucine arylamidase and naphthol-AS-BIphosphohydrolase. Activities of esterase (C4), esterase lipase (C8) and acid phosphatase are positive for all three strains, but only weakly positive for W. thalassocola KCTC 12221T. All three strains are negative for the following: anaerobic growth; Gram-staining; nitrate reduction; H2S production; production of flexirubin-type pigments; hydrolysis of urea, hypoxanthine and xanthine; acid production from L-arabinose, D-fructose, D-galactose, lactose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, trehalose, D-xylose, myo-inositol, Dmannitol and D-sorbitol; susceptibility to gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activities of lipase (C14), a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase.
The predominant isoprenoid quinone detected in strain WD-2-2T was menaquinone-6 (MK-6), in line with other members of the genus Winogradskyella (Nedashkovskaya et al., 2005, 2012; Begum et al., 2013) and all other members of the family Flavobacteriaceae (Bernardet, 2011). The cellular fatty acid profiles of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola are compared in Table 2. The major fatty acids (.10 % of the total fatty acids) detected in strain WD-2-2T were iso-C15 : 1 G (21.8 %), iso-C17 : 0 3-OH (21.5 %) and iso-C15 : 0 (15.9 %). The fatty acid profiles of the three strains were similar, although there were differences in the proportions of some fatty acids and in the presence or absence of some fatty acids (Table 2). The major polar lipids detected in strain WD-2-2T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid (Fig. S1, available in the online Supplementary Material). The polar lipid profile of strain WD-2-2T was similar to those of W. litorisediminis DPS-8T and W. thalassocola KCTC 12221T in that the only identified phospholipid is phosphatidylethanolamine, and one unidentified lipid is a major polar lipid, although they were distinguishable from each other by the presence or absence of several unidentified lipids (Lee et al., 2012; Kang et al., 2013). The DNA G+C content of strain WD-2-2T was 36.4 mol%, a value slightly higher than that of W. litorisediminis DPS-8T but in the range reported for members of the genus Winogradskyella (Table 1; Kang et al., 2013; Begum et al., 2013). The results obtained from the phylogenetic and chemotaxonomic analyses are sufficient to determine the taxonomic position of strain WD2-2T as a member of the genus Winogradskyella.
Characteristic Growth at: 4 and 10 uC 37 and 40 uC Hydrolysis of: Aesculin Starch L-Tyrosine Acid production from: Cellobiose Sucrose Susceptibility to: Ampicillin Cephalothin Novobiocin Penicillin G Tetracycline Enzyme activity (API ZYM) Valine arylamidase Cystine arylamidase Trypsin a-Chymotrypsin DNA G+C content (mol%)
1
2
3
2 +
+ 2
+* 2*
2 2 +
+ + 2
+ 2 +
2 2
2 +
+ +
+ + + + 2
2 2 + 2 +
2 + 2 2 2
+ + + + 36.4
+ + + 2 34.7
2 2 2 2 34.6*
*Data taken from Nedashkovskaya et al. (2005).
Mean DNA–DNA relatedness between strain WD-2-2T and W. litorisediminis DPS-8T was 13 %. Strain WD-2-2T was distinguished from the type strains of W. litorisediminis and W. thalassocola by differences in several phenotypic characteristics, including the temperature for growth, hydrolysis of some substrates, acid production from some substrates, susceptibility to some antibiotics and activities of some enzymes (Table 1). These differences, in combination with the phylogenetic and genetic distinctiveness of strain WD-2-2T, suggest that the novel strain is separate from other species of the genus Winogradskyella (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, strain WD-2-2T is therefore considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella wandonensis sp. nov. is proposed. Description of Winogradskyella wandonensis sp. nov.
litorisediminis with a bootstrap resampling value of 96.7 % (Fig. 1). The relationship between strain WD-2-2T and W. litorisediminis DPS-8T was maintained in trees reconstructed using the maximum-likelihood and maximumparsimony algorithms (Fig. 1). Strain WD-2-2T exhibited 16S rRNA gene sequence similarity of 97.4 % to W. 1522
Winogradskyella wandonensis (wan.do.nen9sis. N.L. fem. adj. wandonensis pertaining to Wando, an island of South Korea, where the type strain was isolated). Cells are Gram-stain-negative, non-flagellated, non-gliding and rod-shaped, approximately 0.2–0.5 mm wide and International Journal of Systematic and Evolutionary Microbiology 64
Winogradskyella wandonensis sp. nov.
Winogradskyella undariae WS-MY5T (KC261665)
58.6
0.01
Winogradskyella pacifica KMM 6019T (GQ181061) Winogradskyella psychrotolerans RS-3T (FN377721) Winogradskyella thalassocola KMM 3907T (AY521223) Winogradskyella rapida SCB 36T (U64013) 100 Winogradskyella arenosi R 60T (AB438962)
72.7
Winogradskyella damuponensis F081-2T (HQ336488) Winogradskyella eximia KMM 3944T (AY521225) Winogradskyella multivorans T-Y1T (JQ354979) Winogradskyella lutea A73T (FJ919968)
59.7
Winogradskyella epiphytica KMM 3906T (AY521224) 98.7 79.4
69.2
Winogradskyella pulchriflava EM106T (JN896598) Winogradskyella echinorum KMM 6211T (EU727254) Winogradskyella ulvae KMM 6390T (HQ456127)
97.4 87.5
Winogradskyella aquimaris DPG-24T (HM368527) Winogradskyella poriferorum UST030701-295T (AY848823)
Winogradskyella exilis 022-2-26T (FJ595484) Winogradskyella wandonensis WD-2-2T (KF768343)
96.7
Winogradskyella litorisediminis DPS-8T (JQ432561) Marinivirga aestuarii KYW371T (HQ405792)
84.1 76.7
Pontirhabdus pectinovorans JC2675T (HM475134) Postechiella marina M091T (HQ336487) Lacinutrix copepodicola DJ3T (AY694001) Lacinutrix himadriensis E4-9aT (FN377744)
100
Gaetbulibacter saemankumensis SMK-12T (AY883937)
62.9 58.5
Gaetbulibacter marinus IMCC1914T (EF108219) Meridianimaribacter flavus NH57NT (FJ360684) 100
Mariniflexile gromovii KMM 6038T (DQ312294) Mariniflexile fucanivorans SW5T (AJ628046) Flaviramulus basaltis H35T (DQ361033) Psychroserpens burtonensis ACAM 188T (U62913)
100
Psychroserpens damuponensis F051-1T (HQ336490) Capnocytophaga ochracea ATCC 27872T (U41350)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Winogradskyella wandonensis sp. nov. WD-2-2T and representatives of other species of the genus Winogradskyella and of some other related taxa. Only bootstrap values greater than 50 % (expressed as percentages of 1000 replications) are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in trees reconstructed with the maximum-likelihood and maximum-parsimony algorithms. Capnocytophaga ochracea ATCC 27872T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
0.7–10.0 mm long. Colonies on MA are circular, slightly convex, smooth, glistening, strong orange–yellow and 1.0– 1.5 mm in diameter after incubation for 3 days at 30 uC. The optimal temperature for growth is 30 uC; growth occurs at 15 and 40 uC but not at 10 or 45 uC. Optimal growth occurs at pH 7.0–8.0; growth occurs at pH 6.0 but not at pH 5.5. Optimal growth occurs in the presence of approximately 2.0 % (w/v) NaCl; growth occurs in the presence of 0.5–5.0 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Flexirubin-type pigments are not produced. H2S is not produced. Casein, gelatin, Tweens 20, 40, 60 and 80 and http://ijs.sgmjournals.org
L-tyrosine
are hydrolysed, but aesculin, hypoxanthine, starch, urea and xanthine are not. Acid is produced from D-glucose, maltose and D-mannose, but not from Larabinose, cellobiose, D-fructose, D-galactose, lactose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, sucrose, trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. In assays with the API ZYM system, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase and naphthol-AS-BIphosphohydrolase are present, but activities of lipase (C14), a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, 1523
S. Park and others
Table 2. Cellular fatty acid compositions of strain WD-2-2T and the type strains of W. litorisediminis and W. thalassocola T
T
Strains: 1, WD-2-2 ; 2, W. litorisediminis DPS-8 ; 3, W. thalassocola KCTC 12221T (data from Park & Yoon, 2013). Data were obtained from this study unless indicated. Values are percentages of total fatty acids; fatty acids that represented ,0.5 % in all strains were omitted. TR, Trace (,0.5 %); 2, not detected. Fatty acid Straight-chain C14 : 0 C16 : 0 C18 : 0 Branched iso-C14 : 0 iso-C15 : 0 iso-C15 : 1 G* anteiso-C15 : 0 anteiso-C15 : 1 A* iso-C16 : 0 iso-C16 : 1 G* iso-C16 : 1 H* Hydroxy C15 : 0 2-OH C15 : 0 3-OH C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH iso-C13 : 0 3-OH iso-C14 : 0 3-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Unsaturated C15 : 1v6c C17 : 1v6c C18 : 1v5c iso-C17 : 1v9c Summed features Summed feature 3D
1
2
3
Acknowledgements This work was supported by the Program for Collection, Management and Utilization of Biological Resources from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (grant NRF2013M3A9A5075953).
References Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel’s Manual for
the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. 0.9 2
2 0.9 2
1.1 1.5 0.7
0.8 15.9 21.8 1.3 1.6 1.4 1.9 2
0.8 17.2 25.0 2.2 1.7 2.0 0.8 2
1.3 17.7 15.0 11.4 2.6 1.2 2 2.2
1.2 0.7 1.8 0.9 0.8
0.9 0.7 1.1 1.1 0.9 0.8
1.4 1.5 0.8 2.6 2
TR
TR
TR
Begum, Z., Srinivas, T. N. R., Manasa, P., Sailaja, B., Sunil, B., Prasad, S. & Shivaji, S. (2013). Winogradskyella psychrotolerans sp.
nov., a marine bacterium of the family Flavobacteriaceae isolated from Arctic sediment. Int J Syst Evol Microbiol 63, 1646–1652. Bernardet, J.-F. (2011). Family I. Flavobacteriaceae Reichenbach 1992. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 4, pp. 106–111. Edited by N. R. Krieg, W. Ludwig, W. B. Whitman, B. P. Hedlund, B. J. Paster, J. T. Staley, N. Ward, D. Brown & A. Parte. New York: Springer. Bernardet, J.-F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002).
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861– 1868.
0.6 7.8 8.1 21.5
6.9 5.7 19.9
2 10.8 10.4 8.4
2 0.7 2 2
2 2 2 2
2.1 0.7 0.9 2.1
9.8
10.8
3.0
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
*Double bond position indicated by a capital letter is unknown. DSummed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Ivanova, E. P., Christen, R., Gorshkova, N. M., Zhukova, N. V., Kurilenko, V. V., Crawford, R. J. & Mikhailov, V. V. (2010).
a-mannosidase and a-fucosidase are absent. Susceptible to
Winogradskyella exilis sp. nov., isolated from the starfish Stellaster equestris, and emended description of the genus Winogradskyella. Int J Syst Evol Microbiol 60, 1577–1580.
TR
ampicillin, carbenicillin, cephalothin, chloramphenicol, lincomycin, novobiocin, oleandomycin and penicillin G, but not to gentamicin, kanamycin, neomycin, polymyxin B, streptomycin or tetracycline. The predominant menaquinone is MK-6. The major fatty acids (.10 % of the total fatty acids) are iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0. The polar lipids are phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid. The type strain, WD-2-2T (5KCTC 32579T5CECT 8445T), was isolated from a tidal flat at Wando in the South Sea of South Korea. The DNA G+C content of the type strain is 36.4 mol%. 1524
Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Euze´by, J. P. (1997). List of Bacterial Names with Standing in
Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 47, 590–592. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
Kang, C.-H., Lee, S.-Y. & Yoon, J.-H. (2013). Winogradskyella
litorisediminis sp. nov., isolated from coastal sediment. Int J Syst Evol Microbiol 63, 1793–1799. Kim, S. B. & Nedashkovskaya, O. I. (2010). Winogradskyella pacifica
sp. nov., a marine bacterium of the family Flavobacteriaceae. Int J Syst Evol Microbiol 60, 1948–1951. Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207. La´nyi, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67. Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Dobretsov, S., Lau, K. W. K., Wu, M., Wong, P.-K., Pawlik, J. R. & Qian, P.-Y. (2005).
Winogradskyella poriferorum sp. nov., a novel member of the family International Journal of Systematic and Evolutionary Microbiology 64
Winogradskyella wandonensis sp. nov. Flavobacteriaceae isolated from a sponge in the Bahamas. Int J Syst Evol Microbiol 55, 1589–1592.
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
Lee, S.-Y., Park, S., Oh, T.-K. & Yoon, J.-H. (2012). Winogradskyella
2nd edn, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
aquimaris sp. nov., isolated from seawater. Int J Syst Evol Microbiol 62, 1814–1818.
Romanenko, L. A., Tanaka, N., Frolova, G. M. & Mikhailov, V. V. (2009). Winogradskyella arenosi sp. nov., a member of the family
Lee, D.-H., Cho, S. J., Kim, S. M. & Lee, S. B. (2013). Winogradskyella
Flavobacteriaceae isolated from marine sediments from the Sea of Japan. Int J Syst Evol Microbiol 59, 1443–1446.
damuponensis sp. nov., isolated from seawater. Int J Syst Evol Microbiol 63, 321–326.
Sasser, M. (1990). Identification of bacteria by gas chromatography of
Leifson, E. (1963). Determination of carbohydrate metabolism of
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI, Inc.
marine bacteria. J Bacteriol 85, 1183–1184.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977).
DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846– 849.
Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol 27, 104–117. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Snauwaert, C., Vancanneyt, M., Swings, J., Kim, K.-O., Lysenko, A. M., Rohde, M. & other authors (2005). Winogradskyella thalassocola gen. nov., sp.
nov., Winogradskyella epiphytica sp. nov. and Winogradskyella eximia sp. nov., marine bacteria of the family Flavobacteriaceae. Int J Syst Evol Microbiol 55, 49–55. Nedashkovskaya, O. I., Vancanneyt, M., Kim, S. B. & Zhukova, N. V. (2009). Winogradskyella echinorum sp. nov., a marine bacterium of the
family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius. Int J Syst Evol Microbiol 59, 1465–1468. Nedashkovskaya, O. I., Kukhlevskiy, A. D. & Zhukova, N. V. (2012).
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. Yoon, J.-H. & Lee, S.-Y. (2012). Winogradskyella multivorans sp. nov.,
a polysaccharide-degrading bacterium isolated from seawater of an oyster farm. Antonie van Leeuwenhoek 102, 231–238. Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of
Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Winogradskyella ulvae sp. nov., an epiphyte of a Pacific seaweed, and emended descriptions of the genus Winogradskyella and Winogradskyella thalassocola, Winogradskyella echinorum, Winogradskyella exilis and Winogradskyella eximia. Int J Syst Evol Microbiol 62, 1450–1456.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
Park, S. & Yoon, J.-H. (2013). Winogradskyella undariae sp. nov., a
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
member of the family Flavobacteriaceae isolated from a brown algae reservoir. Antonie van Leeuwenhoek 104, 619–626. Pinhassi, J., Nedashkovskaya, O. I., Hagstro¨m, A. & Vancanneyt, M. (2009). Winogradskyella rapida sp. nov., isolated from protein-
enriched seawater. Int J Syst Evol Microbiol 59, 2180–2184.
http://ijs.sgmjournals.org
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194. Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp.
Yoon, B.-J., Byun, H.-D., Kim, J.-Y., Lee, D.-H., Kahng, H.-Y. & Oh, D.-C. (2011). Winogradskyella lutea sp. nov., isolated from seawater,
and emended description of the genus Winogradskyella. Int J Syst Evol Microbiol 61, 1539–1543.
1525
