International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1876–1881
DOI 10.1099/ijs.0.060533-0
Seonamhaeicola aphaedonensis gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a tidal flat sediment Sooyeon Park,1 Sung-Min Won,1 Doo-Sang Park2 and Jung-Hoon Yoon1 Correspondence
1
Jung-Hoon Yoon [email protected]
2
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yuseong, Daejeon, Republic of Korea
A Gram-stain-negative, non-motile, rod-shaped bacterial strain, AH-M5T, which was isolated from a tidal flat sediment at Aphae Island in South Korea, was characterized taxonomically. Strain AHM5T grew optimally at 25 6C, at pH 7.0–8.0 and in presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain AH-M5T clustered coherently with the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus, showing 93.4– 94.3 % sequence similarity. The novel strain exhibited 16S rRNA gene sequence similarity values of less than 93.4 % to the type strains of other recognized species. Strain AH-M5T contained MK6 as the predominant menaquinone and iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) as the major fatty acids. The polar lipid profile of strain AH-M5T containing phosphatidylethanolamine and one unidentified lipid as major components was differentiated from those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus. The DNA G+C content of strain AH-M5T was 34.8 mol%. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, demonstrated that strain AH-M5T is distinguished from Mangrovimonas yunxiaonensis and Meridianimaribacter flavus. On the basis of the data presented, strain AH-M5T is considered to represent a novel genus and species within the family Flavobacteriaceae, for which the name Seonamhaeicola aphaedonensis gen. nov., sp. nov. is proposed. The type strain of the type species is AH-M5T (5KCTC 32578T5CECT 8487T).
A variety of tidal flats on western and southern coasts of the Korean peninsula have been proven to be good environments from which to isolate novel bacterial taxa belonging to the family Flavobacteriaceae (Jung et al., 2005, 2013; Kwon et al., 2006; Kim et al., 2008; Yoon et al., 2008, 2013; Yi & Chun, 2011; Lee et al., 2012; Jeong et al., 2013; Park et al., 2013). A tidal flat in Aphae Island, located in the south-western sea of the Korean peninsula, has been used as a new site to screen for novel bacteria. One of bacterial strains isolated, designated AH-M5T, which belongs to the family Flavobacteriaceae, is described in this study. Comparative 16S rRNA gene sequence analysis indicated that the novel strain is phylogenetically most closely affiliated to the genera Meridianimaribacter and Mangrovimonas, members of the family Flavobacteriaceae. The genus Meridianimaribacter was proposed by Wang et al. The GenBank/EMBL/DDBJ accession number for the16S rRNA gene sequence of strain AH-M5T is KF876013. Two supplementary figures are available with the online version of this paper.
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(2010) and at the time of writing comprises a single species with validly published name, Meridianimaribacter flavus. The genus Mangrovimonas was recently proposed by Li et al. (2013) with the description of a single novel species, Mangrovimonas yunxiaonensis. Members of the two genera have been isolated from mangrove and marine sediments (Wang et al., 2010; Li et al., 2013). The aim of the present work was to determine the exact taxonomic position of strain AH-M5T by using a polyphasic characterization. A tidal flat sediment was collected from Aphae Island in the south-western sea, South Korea, and used as a source for the isolation of bacterial strains. Strain AH-M5T was isolated by the standard dilution plating technique on marine agar 2216 (MA; Becton Dickinson) at 25 uC and cultivated routinely under the same conditions. Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T, which were used for reference strains for the analyses of fatty acids and polar lipids and other phenotypic characterization tests, were obtained from the Laboratorium voor Microbiologie Universiteit Gent (LMG), Gent, Belgium 060533 G 2014 IUMS Printed in Great Britain
Seonamhaeicola aphaedonensis gen. nov., sp. nov.
and the Korean Agricultural Culture Collection (KACC), Suwon, South Korea, respectively. 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, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the 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 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 25 uC. Growth at 4, 10, 15, 20, 25, 30 and 37 uC was measured on MA 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 sodium carbonate buffers; the pH of the medium was verified after autoclaving. Growth in the absence of NaCl and in the presence of 0.5, 1.0, 2.0 and 3.0 % (w/v) NaCl was investigated in trypticase soy broth prepared according to the formula of the BD medium, using supplementation with 0.45 % (w/v) MgCl2 . 6H2O and without MgCl2 . 6H2O, except that NaCl was excluded. Growth in the presence of 2.0–8.0 % NaCl (in increments of 1.0 %) was investigated in MB. 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 by La´nyi (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were 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. Degradation of CMcellulose (Sigma) was tested on basal medium agar [12 g gellan gum, 1 g yeast extract, 0.5 g NH4Cl, 50 ml 1M Tris/ HCl (pH 7.4) and 5 g low melting agarose per litre artificial seawater] containing 0.5 % (w/v) CM-cellulose. Degradation of CM-cellulose was revealed by flooding the agar with a 0.1 % (w/v) Congo red aqueous solution. 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 http://ijs.sgmjournals.org
(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, after incubation for 8 h at 25 uC, by using the API ZYM system (bioMe´rieux). Cell biomass of strain AH-M5T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in MB at 25 uC, and cell biomass of Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T for polar lipid analysis was obtained from cultures grown for 3 days in MB at 30 uC. Chromosomal DNA was extracted and purified according to the method described by Yoon et al. (1996), with the modification that RNase T1 was used in combination with RNase A to minimize contamination of 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). Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987), using reversedphase HPLC with a YMC ODS-A (25064.6 mm) column. The isoprenoid quinones were eluted by a mixture of methanol/2-propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm. For cellular fatty acid analysis, cell masses of strain AH-M5T, Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T were harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell masses was standardized by observing the growth development of colonies on the 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 (6890; Hewlett Packard) and identified by 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 with molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol reagents (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 with a YMC ODS-A (25064.6 mm) column. The nucleotides 1877
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were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm. Morphological, cultural, physiological and biochemical characteristics of strain AH-M5T are given in the genus and species descriptions or in Table 1 and Fig. S1 (available in the online Supplementary Material). The almost-complete 16S rRNA gene sequence of strain AH-M5T comprised 1440 nt, approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain AHM5T clustered with the type strain of Mangrovimonas yunxiaonensis by a bootstrap resampling value of 77.3 %, and this cluster joined the type strain of Meridianimaribacter flavus by a bootstrap resampling value of 53.2 % (Fig. 1). The relationships among these three strains were also maintained in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain AHM5T exhibited 16S rRNA gene sequence similarity values of 94.3 and 93.4 % to the type strains of Meridianimaribacter flavus and Mangrovimonas yunxiaonensis, respectively, and of less than 93.4 % to the type strains of other recognized species. The predominant isoprenoid quinone detected in strain AH-M5T was menaquinone-6 (MK-6), which is compatible with all members of the family Flavobacteriaceae (Bernardet, 2011). In Table 2, the fatty acid profile of strain AH-M5T is compared with those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus, grown and analysed under identical conditions in this study. The major fatty acids (.10 % of the total fatty acids) found in strain AH-M5T were iso-C15 : 1 G (17.9 %), iso-C15 : 0 (15.8 %), isoC17 : 0 3-OH (15.4 %) and summed feature 3 (C16 : 1v7c and/ or C16 : 1v6c, 11.2 %). The fatty acid profile of strain AHM5T was similar to those of Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T, but the profiles were distinguishable by differences in the proportions of some fatty acids (Table 2). In particular, the fatty acid profile of strain AH-M5T was distinguished from Mangrovimonas yunxiaonensis LMG 27142T by differences in the proportions of iso-C15 : 0 and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids detected in strain AH-M5T were phosphatidylethanolamine and one unidentified lipid (Fig. S2). The polar lipid profile of strain AH-M5T was distinguishable from those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus analysed under identical conditions in this study. In particular, the polar lipid profile of strain AH-M5T was distinguished from Mangrovimonas yunxiaonensis LMG 27142T by the absence of additional unidentified lipids, and from Meridianimaribacter flavus KACC 15613T by not having one additional unidentified aminolipid (AL2) and by having phosphatidylethanolamine as a major component. The DNA G+C content of strain AHM5T was 34.8 mol%, a value lower than that of the type strain of Mangrovimonas yunxiaonensis and little higher 1878
Table 1. Differential phenotypic characteristics of strain AHM5T and the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus Strains: 1, AH-M5T; 2, Mangrovimonas yunxiaonensis LMG 27142T; 3, Meridianimaribacter flavus KACC 15613T. Data obtained from this study unless indicated otherwise. All strains are positive for catalase and oxidase activities*; hydrolysis of starch* (weak for two reference strains); susceptibility to carbenicillin, cephalothin, chloramphenicol, lincomycin, oleandomycin, penicillin G and tetracycline; and activity of alkaline phosphatase and leucine arylamidase. All strains are negative for anaerobic growth*; Gram-staining; nitrate reduction*; H2S production*; acid production from L-arabinose, D-fructose, lactose, melezitose, melibiose, raffinose, D-ribose, trehalose, myoinositol, D-mannitol and D-sorbitol; susceptibility to gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activity of lipase (C14), cystine arylamidase, trypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative; W, weakly positive. Characteristic Growth at:* 10 uC 37 uC Gliding motility* NaCl range (%) for growth* Hydrolysis of:* Aesculin Casein Gelatin Tween 80 Urea Acid production from: Cellobiose D-Galactose D-Glucose Maltose Mannose L-Rhamnose Sucrose D-Xylose Susceptibility to: Ampicillin Novobiocin Enzyme activity (API ZYM) Esterase (C4) Esterase lipase (C8) Valine arylamidase a-Chymotrypsin Acid phosphatase Naphthol-AS-BIphosphohydrolase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1 2 2 2 1.0–5.0
2
3
+ + + + + + 1.0–7.0 0.5–4.0
2 + 2 + 2
+
+ 2 + 2 2
2 + + 2 + + + +
2 2 2 2 2 2 2 2
+ + + + + 2 2 2
+ +
2 +
2 2
2 2 2 2 2 2
+ + + 2 + +
+ + + + + +
+ 34.8
2 38.6
2 32.7
W
2 + W
*Data for Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T taken from Li et al. (2013) and Wang et al. (2010), respectively.
International Journal of Systematic and Evolutionary Microbiology 64
Seonamhaeicola aphaedonensis gen. nov., sp. nov.
Gaetbulibacter marinus IMCC1914T (EF108219)
96.4
0.01
Gaetbulibacter lutimaris D1-y4T (JF739861) Gaetbulibacter saemankumensis SMK-12T (AY883937)
63.2
Gaetbulibacter aestuarii KYW382T (GU552681) Siansivirga zeaxanthinifaciens CC-SAMT-1T (HM179539)
95.5
Tamlana agarivorans JW-26T (EU221275) Tamlana crocina HST1-43T (AM286230)
68.9
Aestuariibaculum suncheonense SC17T (JF751043) Snuella lapsa JC2132T (HM475133) Seonamhaeicola aphaedonensis AH-M5T (KF876013)
77.3
Mangrovimonas yunxiaonensis LYYY01T (JQ937283)
53.2
Meridianimaribacter flavus NH57NT (FJ360684) Winogradskyella thalassocola KMM 3907T (AY521223)
86.3
Winogradskyella arenosi R60T (AB438962)
56.4 60.6
87.8
86.7
Winogradskyella echinorum KMM 6211T (EU727254) Winogradskyella ulvae KMM 6390T (HQ456127) Winogradskyella litorisediminis DPS-8T (JQ432561) Winogradskyella aquimaris DPG-24T (HM368527)
100
Winogradskyella poriferorum UST030701-295T (AY848823) Jejuia pallidilutea EM39T (EU443204)
99.7 100
Hyunsoonleella jejuensis CNU004T (EF217419) Bizionia echini KMM 6177T (FJ716799) Bizionia hallyeonensis T-y7T (JN885199) Bizionia paragorgiae KMM 6029T (AY651070) Formosa algae KMM 3553T (AY228461)
100 79.1
99.5
Formosa agariphila KMM 3901T (AY187688) Flavivirga jejuensis JC2682T (HM475139) Flavivirga amylovorans JC2681T (HM475138)
53.8 88.8
Psychroserpens mesophilus KOPRI 13649T (DQ001321) Psychroserpens burtonensis ACAM 188T (U62913) Capnocytophaga ochracea ATCC 27872T (U41350)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain AH-M5T and representatives of some other related taxa. Only bootstrap values (expressed as percentages of 1000 replications) .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. Capnocytophaga ochracea ATCC 27872T (GenBank accession no. U41350) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
than that of the type strain of Meridianimaribacter flavus (Table 1). T
Strain AH-M5 was differentiated from the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus by differences in phenotypic characteristics, including growth at 10 and 37 uC, gliding motility, the NaCl range for growth, hydrolysis of some substrates, acid production from substrates and activity of some enzymes (Table 1). The phylogenetic analysis and differential chemotaxonomic data and other phenotypic properties suggest that strain AH-M5T represents a novel species in a new genus within the family Flavobacteriaceae, for which the name Seonamhaeicola aphaedonensis gen. nov., sp. nov. is proposed. http://ijs.sgmjournals.org
Description of Seonamhaeicola gen. nov. Seonamhaeicola [Se.o.nam.hae.i9co.la. N.L. n. Seonamhae the Korean name of the south-west sea in South Korea; L. suff. -cola (from L. n. incola) a dweller, inhabitant; N.L. masc. n. Seonamhaeicola a dweller of the south-west sea in South Korea]. Cells are Gram-stain-negative, aerobic, non-flagellated, non-gliding rods. Catalase- and oxidase-positive. Nitrate reduction is negative. The predominant menaquinone is MK-6. The major fatty acids (.10 % of the total fatty acids) are iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids are phosphatidylethanolamine and one 1879
S. Park and others
Table 2. Cellular fatty acid contents (%) of strain AH-M5T and the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus Strains: 1, AH-M5T; 2, Mangrovimonas yunxiaonensis LMG 27142T; 3, Meridianimaribacter flavus KACC 15613T. All data obtained from this study. Fatty acids that represented ,0.5 % in all strains were omitted. TR, Traces (,0.5 %); 2, Not detected. Fatty acid Straight-chain fatty acid C14 : 0 C16 : 0 Branched fatty acid iso-C13 : 0 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-C17 : 0 iso-C17 : 1v9c Hydroxy fatty acid C15 : 0 2-OH C15 : 0 3-OH C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Summed featureD 3
1
2
3
2 0.9
0.7 1.5
3.2
TR
0.8 1.2 30.5 21.7 1.8 1.0 3.2 1.5 0.7 2.0
1.4 15.8 17.9 7.0 2.6 1.7 0.8 2 2 1.7
TR
TR
0.7 1.2 0.8
TR
TR
0.7 22.9 20.7 1.0 TR
1.6 TR TR
2
TR
TR
9.7 7.6 15.4
4.3 4.0 19.4
0.6 0.7 2.3 0.7 0.7 6.6 8.8 19.5
11.2
1.0
7.1
TR
3.5
*Double bond position indicated by a capital letter is unknown. DSummed features are groups of two or three fatty acids that could not be separated by the Microbial Identification System. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Optimal growth temperature is 25 uC; growth occurs at 15 and 35 uC, but not at 10 or 37 uC. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of 2.0 % (w/v) NaCl; growth occurs in the presence of 1.0–5.0 % (w/v) NaCl. Mg2+ ions are required for growth. H2S is not produced. Flexirubin-type pigments are not produced. Casein, starch and Tweens 20, 40, 60 and 80 are hydrolysed, but aesculin, CM-cellulose, gelatin, hypoxanthine, L-tyrosine, urea and xanthine are not. Acid is produced from Dgalactose, D-glucose, D-mannose, L-rhamnose, sucrose and D-xylose, but not from L-arabinose, cellobiose, D-fructose, lactose, maltose, melezitose, melibiose, raffinose, D-ribose, trehalose, myo-inositol, D-mannitol or D-sorbitol. Susceptible to ampicillin, carbenicillin, cephalothin, chloramphenicol, lincomycin, novobiocin, oleandomycin, penicillin G and tetracycline, but not to gentamicin, kanamycin, neomycin, polymyxin B or streptomycin. In the API ZYM system, alkaline phosphatase, leucine arylamidase and N-acetylb-glucosaminidase activities are present, but esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohdrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, amannosidase and a-fucosidase activities are absent. The type strain, AH-M5T (5KCTC 32578T5CECT 8487T), was isolated from a tidal flat sediment of Aphaedo, an island in the south-west sea, South Korea. The DNA G+C content of the type strain is 34.8 mol%.
ACKNOWLEDGEMENTS This work was supported by the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea Program for Collection, Management and Utilization of Biological Resources (grant NRF2013M3A9A5075953). We are grateful to the KACC for kindly providing Meridianimaribacter flavus KACC 15613T.
REFERENCES Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel’s
unidentified lipid. The DNA G+C content is 34.8 mol%. The type species is Seonamhaeicola aphaedonensis. A member of the family Flavobacteriaceae, phylum Bacteroidetes, according to 16S rRNA gene sequence analysis. Description of Seonamhaeicola aphaedonensis sp. nov. Seonamhaeicola aphaedonensis (ap.hae.do.nen9sis. N.L. masc. adj. aphaedonensis pertaining to Aphaedo, an island of South Korea, from where the type strain was isolated). Exhibits the following properties in addition to those given in the genus description. Cells are 0.2–0.7 mm in diameter and 0.9–8.5 mm in length. Colonies on MA agar are circular, slightly convex, smooth, glistening, strong orange–yellow in colour and 0.5–1.0 mm after incubation for 5 days at 25 uC. 1880
Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. 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, J. T. Staley, D. R. Brown, B. P. Hedlund, B. J. Paster, N. L. Ward, W. Ludwig & W. B. Whitman. 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. International Journal of Systematic and Evolutionary Microbiology 64
Seonamhaeicola aphaedonensis gen. nov., sp. nov. 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.
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.
Jeong, S. H., Park, M. S., Jin, H. M., Lee, K., Park, W. & Jeon, C. O. (2013). Aestuariibaculum suncheonense gen. nov., sp. nov., a marine
Park, S. C., Choe, H. N., Hwang, Y. M., Baik, K. S., Kim, S. N., Lee, Y. S., Jung, J. S. & Seong, C. N. (2013). Marinivirga aestuarii gen.
bacterium of the family Flavobacteriaceae isolated from a tidal flat and emended descriptions of the genera Gaetbulibacter and Tamlana. Int J Syst Evol Microbiol 63, 332–338.
nov., sp. nov., a member of the family Flavobacteriaceae, isolated from marine environments, and emended descriptions of the genera Hyunsoonleella, Jejuia and Pontirhabdus and the species Hyunsoonleella jejuensis, Jejuia pallidilutea and Pontirhabdus pectinivorans. Int J Syst Evol Microbiol 63, 1524–1531.
Jung, S.-Y., Kang, S.-J., Lee, M.-H., Lee, S.-Y., Oh, T.-K. & Yoon, J.-H. (2005). Gaetbulibacter saemankumensis gen. nov., sp. nov., a
novel member of the family Flavobacteriaceae isolated from a tidal flat sediment in Korea. Int J Syst Evol Microbiol 55, 1845–1849. Jung, Y.-T., Lee, J.-S. & Yoon, J.-H. (2013). Hwangdonia seohaensis
gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 3186–3191. Kim, J.-H., Kim, K.-Y., Hahm, Y.-T., Kim, B.-S., Chun, J. & Cha, C.-J. (2008). Actibacter sediminis gen. nov., sp. nov., a marine bacterium of
the family Flavobacteriaceae isolated from tidal flat sediment. Int J Syst Evol Microbiol 58, 139–143. Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in
bacterial systematics. Methods Microbiol 19, 161–207. Kwon, K. K., Lee, H.-S., Jung, H.-B., Kang, J.-H. & Kim, S.-J. (2006).
Yeosuana aromativorans gen. nov., sp. nov., a mesophilic marine bacterium belonging to the family Flavobacteriaceae, isolated from estuarine sediment of the South Sea, Korea. Int J Syst Evol Microbiol 56, 727–732. La´nyi, B. (1987). Classical and rapid identification methods for
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
2nd edn, vol. 4, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Sasser, M. (1990). Identification of bacteria by gas chromatography
of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128. Wang, B., Sun, F., Du, Y., Liu, X., Li, G., Lai, Q., Luo, J. & Shao, Z. (2010). Meridianimaribacter flavus gen. nov., sp. nov., a member of
the family Flavobacteriaceae isolated from marine sediment of the South China Sea. Int J Syst Evol Microbiol 60, 121–127. Yi, H. & Chun, J. (2011). Snuella lapsa gen. nov., sp. nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 61, 2469–2474. 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
medically important bacteria. Methods Microbiol 19, 1–67.
Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Lee, S.-Y., Lee, M.-H., Oh, T.-K. & Yoon, J.-H. (2012). Lutibacter
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
aestuarii sp. nov., isolated from a tidal flat sediment, and emended description of the genus Lutibacter Choi and Cho 2006. Int J Syst Evol Microbiol 62, 420–424. Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J Bacteriol 85, 1183–1184. Li, Y., Bai, S., Yang, C., Lai, Q., Zhang, H., Chen, Z., Wei, J., Zheng, W., Tian, Y. & Zheng, T. (2013). Mangrovimonas yunxiaonensis gen. nov.,
sp. nov., isolated from mangrove sediment. Int J Syst Evol Microbiol 63, 2043–2048. Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977).
Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol 27, 104–117.
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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.
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454. Yoon, J.-H., Kang, S.-J., Jung, Y.-T. & Oh, T.-K. (2008). Aestuariicola saemankumensis gen. nov., sp. nov., a member of the family Flavobacteriaceae, isolated from tidal flat sediment. Int J Syst Evol Microbiol 58, 2126–2131. Yoon, J.-H., Lee, S.-Y. & Oh, T.-K. (2013). Gaetbulibacter lutimaris sp. nov., isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 995–1000.
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DOI 10.1099/ijs.0.060533-0
Seonamhaeicola aphaedonensis gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a tidal flat sediment Sooyeon Park,1 Sung-Min Won,1 Doo-Sang Park2 and Jung-Hoon Yoon1 Correspondence
1
Jung-Hoon Yoon [email protected]
2
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yuseong, Daejeon, Republic of Korea
A Gram-stain-negative, non-motile, rod-shaped bacterial strain, AH-M5T, which was isolated from a tidal flat sediment at Aphae Island in South Korea, was characterized taxonomically. Strain AHM5T grew optimally at 25 6C, at pH 7.0–8.0 and in presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain AH-M5T clustered coherently with the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus, showing 93.4– 94.3 % sequence similarity. The novel strain exhibited 16S rRNA gene sequence similarity values of less than 93.4 % to the type strains of other recognized species. Strain AH-M5T contained MK6 as the predominant menaquinone and iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) as the major fatty acids. The polar lipid profile of strain AH-M5T containing phosphatidylethanolamine and one unidentified lipid as major components was differentiated from those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus. The DNA G+C content of strain AH-M5T was 34.8 mol%. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, demonstrated that strain AH-M5T is distinguished from Mangrovimonas yunxiaonensis and Meridianimaribacter flavus. On the basis of the data presented, strain AH-M5T is considered to represent a novel genus and species within the family Flavobacteriaceae, for which the name Seonamhaeicola aphaedonensis gen. nov., sp. nov. is proposed. The type strain of the type species is AH-M5T (5KCTC 32578T5CECT 8487T).
A variety of tidal flats on western and southern coasts of the Korean peninsula have been proven to be good environments from which to isolate novel bacterial taxa belonging to the family Flavobacteriaceae (Jung et al., 2005, 2013; Kwon et al., 2006; Kim et al., 2008; Yoon et al., 2008, 2013; Yi & Chun, 2011; Lee et al., 2012; Jeong et al., 2013; Park et al., 2013). A tidal flat in Aphae Island, located in the south-western sea of the Korean peninsula, has been used as a new site to screen for novel bacteria. One of bacterial strains isolated, designated AH-M5T, which belongs to the family Flavobacteriaceae, is described in this study. Comparative 16S rRNA gene sequence analysis indicated that the novel strain is phylogenetically most closely affiliated to the genera Meridianimaribacter and Mangrovimonas, members of the family Flavobacteriaceae. The genus Meridianimaribacter was proposed by Wang et al. The GenBank/EMBL/DDBJ accession number for the16S rRNA gene sequence of strain AH-M5T is KF876013. Two supplementary figures are available with the online version of this paper.
1876
(2010) and at the time of writing comprises a single species with validly published name, Meridianimaribacter flavus. The genus Mangrovimonas was recently proposed by Li et al. (2013) with the description of a single novel species, Mangrovimonas yunxiaonensis. Members of the two genera have been isolated from mangrove and marine sediments (Wang et al., 2010; Li et al., 2013). The aim of the present work was to determine the exact taxonomic position of strain AH-M5T by using a polyphasic characterization. A tidal flat sediment was collected from Aphae Island in the south-western sea, South Korea, and used as a source for the isolation of bacterial strains. Strain AH-M5T was isolated by the standard dilution plating technique on marine agar 2216 (MA; Becton Dickinson) at 25 uC and cultivated routinely under the same conditions. Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T, which were used for reference strains for the analyses of fatty acids and polar lipids and other phenotypic characterization tests, were obtained from the Laboratorium voor Microbiologie Universiteit Gent (LMG), Gent, Belgium 060533 G 2014 IUMS Printed in Great Britain
Seonamhaeicola aphaedonensis gen. nov., sp. nov.
and the Korean Agricultural Culture Collection (KACC), Suwon, South Korea, respectively. 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, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the 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 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 25 uC. Growth at 4, 10, 15, 20, 25, 30 and 37 uC was measured on MA 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 sodium carbonate buffers; the pH of the medium was verified after autoclaving. Growth in the absence of NaCl and in the presence of 0.5, 1.0, 2.0 and 3.0 % (w/v) NaCl was investigated in trypticase soy broth prepared according to the formula of the BD medium, using supplementation with 0.45 % (w/v) MgCl2 . 6H2O and without MgCl2 . 6H2O, except that NaCl was excluded. Growth in the presence of 2.0–8.0 % NaCl (in increments of 1.0 %) was investigated in MB. 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 by La´nyi (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were 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. Degradation of CMcellulose (Sigma) was tested on basal medium agar [12 g gellan gum, 1 g yeast extract, 0.5 g NH4Cl, 50 ml 1M Tris/ HCl (pH 7.4) and 5 g low melting agarose per litre artificial seawater] containing 0.5 % (w/v) CM-cellulose. Degradation of CM-cellulose was revealed by flooding the agar with a 0.1 % (w/v) Congo red aqueous solution. 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 http://ijs.sgmjournals.org
(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, after incubation for 8 h at 25 uC, by using the API ZYM system (bioMe´rieux). Cell biomass of strain AH-M5T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in MB at 25 uC, and cell biomass of Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T for polar lipid analysis was obtained from cultures grown for 3 days in MB at 30 uC. Chromosomal DNA was extracted and purified according to the method described by Yoon et al. (1996), with the modification that RNase T1 was used in combination with RNase A to minimize contamination of 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). Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987), using reversedphase HPLC with a YMC ODS-A (25064.6 mm) column. The isoprenoid quinones were eluted by a mixture of methanol/2-propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm. For cellular fatty acid analysis, cell masses of strain AH-M5T, Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T were harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell masses was standardized by observing the growth development of colonies on the 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 (6890; Hewlett Packard) and identified by 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 with molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol reagents (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 with a YMC ODS-A (25064.6 mm) column. The nucleotides 1877
S. Park and others
were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm. Morphological, cultural, physiological and biochemical characteristics of strain AH-M5T are given in the genus and species descriptions or in Table 1 and Fig. S1 (available in the online Supplementary Material). The almost-complete 16S rRNA gene sequence of strain AH-M5T comprised 1440 nt, approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain AHM5T clustered with the type strain of Mangrovimonas yunxiaonensis by a bootstrap resampling value of 77.3 %, and this cluster joined the type strain of Meridianimaribacter flavus by a bootstrap resampling value of 53.2 % (Fig. 1). The relationships among these three strains were also maintained in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain AHM5T exhibited 16S rRNA gene sequence similarity values of 94.3 and 93.4 % to the type strains of Meridianimaribacter flavus and Mangrovimonas yunxiaonensis, respectively, and of less than 93.4 % to the type strains of other recognized species. The predominant isoprenoid quinone detected in strain AH-M5T was menaquinone-6 (MK-6), which is compatible with all members of the family Flavobacteriaceae (Bernardet, 2011). In Table 2, the fatty acid profile of strain AH-M5T is compared with those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus, grown and analysed under identical conditions in this study. The major fatty acids (.10 % of the total fatty acids) found in strain AH-M5T were iso-C15 : 1 G (17.9 %), iso-C15 : 0 (15.8 %), isoC17 : 0 3-OH (15.4 %) and summed feature 3 (C16 : 1v7c and/ or C16 : 1v6c, 11.2 %). The fatty acid profile of strain AHM5T was similar to those of Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T, but the profiles were distinguishable by differences in the proportions of some fatty acids (Table 2). In particular, the fatty acid profile of strain AH-M5T was distinguished from Mangrovimonas yunxiaonensis LMG 27142T by differences in the proportions of iso-C15 : 0 and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids detected in strain AH-M5T were phosphatidylethanolamine and one unidentified lipid (Fig. S2). The polar lipid profile of strain AH-M5T was distinguishable from those of the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus analysed under identical conditions in this study. In particular, the polar lipid profile of strain AH-M5T was distinguished from Mangrovimonas yunxiaonensis LMG 27142T by the absence of additional unidentified lipids, and from Meridianimaribacter flavus KACC 15613T by not having one additional unidentified aminolipid (AL2) and by having phosphatidylethanolamine as a major component. The DNA G+C content of strain AHM5T was 34.8 mol%, a value lower than that of the type strain of Mangrovimonas yunxiaonensis and little higher 1878
Table 1. Differential phenotypic characteristics of strain AHM5T and the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus Strains: 1, AH-M5T; 2, Mangrovimonas yunxiaonensis LMG 27142T; 3, Meridianimaribacter flavus KACC 15613T. Data obtained from this study unless indicated otherwise. All strains are positive for catalase and oxidase activities*; hydrolysis of starch* (weak for two reference strains); susceptibility to carbenicillin, cephalothin, chloramphenicol, lincomycin, oleandomycin, penicillin G and tetracycline; and activity of alkaline phosphatase and leucine arylamidase. All strains are negative for anaerobic growth*; Gram-staining; nitrate reduction*; H2S production*; acid production from L-arabinose, D-fructose, lactose, melezitose, melibiose, raffinose, D-ribose, trehalose, myoinositol, D-mannitol and D-sorbitol; susceptibility to gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activity of lipase (C14), cystine arylamidase, trypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative; W, weakly positive. Characteristic Growth at:* 10 uC 37 uC Gliding motility* NaCl range (%) for growth* Hydrolysis of:* Aesculin Casein Gelatin Tween 80 Urea Acid production from: Cellobiose D-Galactose D-Glucose Maltose Mannose L-Rhamnose Sucrose D-Xylose Susceptibility to: Ampicillin Novobiocin Enzyme activity (API ZYM) Esterase (C4) Esterase lipase (C8) Valine arylamidase a-Chymotrypsin Acid phosphatase Naphthol-AS-BIphosphohydrolase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1 2 2 2 1.0–5.0
2
3
+ + + + + + 1.0–7.0 0.5–4.0
2 + 2 + 2
+
+ 2 + 2 2
2 + + 2 + + + +
2 2 2 2 2 2 2 2
+ + + + + 2 2 2
+ +
2 +
2 2
2 2 2 2 2 2
+ + + 2 + +
+ + + + + +
+ 34.8
2 38.6
2 32.7
W
2 + W
*Data for Mangrovimonas yunxiaonensis LMG 27142T and Meridianimaribacter flavus KACC 15613T taken from Li et al. (2013) and Wang et al. (2010), respectively.
International Journal of Systematic and Evolutionary Microbiology 64
Seonamhaeicola aphaedonensis gen. nov., sp. nov.
Gaetbulibacter marinus IMCC1914T (EF108219)
96.4
0.01
Gaetbulibacter lutimaris D1-y4T (JF739861) Gaetbulibacter saemankumensis SMK-12T (AY883937)
63.2
Gaetbulibacter aestuarii KYW382T (GU552681) Siansivirga zeaxanthinifaciens CC-SAMT-1T (HM179539)
95.5
Tamlana agarivorans JW-26T (EU221275) Tamlana crocina HST1-43T (AM286230)
68.9
Aestuariibaculum suncheonense SC17T (JF751043) Snuella lapsa JC2132T (HM475133) Seonamhaeicola aphaedonensis AH-M5T (KF876013)
77.3
Mangrovimonas yunxiaonensis LYYY01T (JQ937283)
53.2
Meridianimaribacter flavus NH57NT (FJ360684) Winogradskyella thalassocola KMM 3907T (AY521223)
86.3
Winogradskyella arenosi R60T (AB438962)
56.4 60.6
87.8
86.7
Winogradskyella echinorum KMM 6211T (EU727254) Winogradskyella ulvae KMM 6390T (HQ456127) Winogradskyella litorisediminis DPS-8T (JQ432561) Winogradskyella aquimaris DPG-24T (HM368527)
100
Winogradskyella poriferorum UST030701-295T (AY848823) Jejuia pallidilutea EM39T (EU443204)
99.7 100
Hyunsoonleella jejuensis CNU004T (EF217419) Bizionia echini KMM 6177T (FJ716799) Bizionia hallyeonensis T-y7T (JN885199) Bizionia paragorgiae KMM 6029T (AY651070) Formosa algae KMM 3553T (AY228461)
100 79.1
99.5
Formosa agariphila KMM 3901T (AY187688) Flavivirga jejuensis JC2682T (HM475139) Flavivirga amylovorans JC2681T (HM475138)
53.8 88.8
Psychroserpens mesophilus KOPRI 13649T (DQ001321) Psychroserpens burtonensis ACAM 188T (U62913) Capnocytophaga ochracea ATCC 27872T (U41350)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain AH-M5T and representatives of some other related taxa. Only bootstrap values (expressed as percentages of 1000 replications) .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. Capnocytophaga ochracea ATCC 27872T (GenBank accession no. U41350) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
than that of the type strain of Meridianimaribacter flavus (Table 1). T
Strain AH-M5 was differentiated from the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus by differences in phenotypic characteristics, including growth at 10 and 37 uC, gliding motility, the NaCl range for growth, hydrolysis of some substrates, acid production from substrates and activity of some enzymes (Table 1). The phylogenetic analysis and differential chemotaxonomic data and other phenotypic properties suggest that strain AH-M5T represents a novel species in a new genus within the family Flavobacteriaceae, for which the name Seonamhaeicola aphaedonensis gen. nov., sp. nov. is proposed. http://ijs.sgmjournals.org
Description of Seonamhaeicola gen. nov. Seonamhaeicola [Se.o.nam.hae.i9co.la. N.L. n. Seonamhae the Korean name of the south-west sea in South Korea; L. suff. -cola (from L. n. incola) a dweller, inhabitant; N.L. masc. n. Seonamhaeicola a dweller of the south-west sea in South Korea]. Cells are Gram-stain-negative, aerobic, non-flagellated, non-gliding rods. Catalase- and oxidase-positive. Nitrate reduction is negative. The predominant menaquinone is MK-6. The major fatty acids (.10 % of the total fatty acids) are iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids are phosphatidylethanolamine and one 1879
S. Park and others
Table 2. Cellular fatty acid contents (%) of strain AH-M5T and the type strains of Mangrovimonas yunxiaonensis and Meridianimaribacter flavus Strains: 1, AH-M5T; 2, Mangrovimonas yunxiaonensis LMG 27142T; 3, Meridianimaribacter flavus KACC 15613T. All data obtained from this study. Fatty acids that represented ,0.5 % in all strains were omitted. TR, Traces (,0.5 %); 2, Not detected. Fatty acid Straight-chain fatty acid C14 : 0 C16 : 0 Branched fatty acid iso-C13 : 0 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-C17 : 0 iso-C17 : 1v9c Hydroxy fatty acid C15 : 0 2-OH C15 : 0 3-OH C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Summed featureD 3
1
2
3
2 0.9
0.7 1.5
3.2
TR
0.8 1.2 30.5 21.7 1.8 1.0 3.2 1.5 0.7 2.0
1.4 15.8 17.9 7.0 2.6 1.7 0.8 2 2 1.7
TR
TR
0.7 1.2 0.8
TR
TR
0.7 22.9 20.7 1.0 TR
1.6 TR TR
2
TR
TR
9.7 7.6 15.4
4.3 4.0 19.4
0.6 0.7 2.3 0.7 0.7 6.6 8.8 19.5
11.2
1.0
7.1
TR
3.5
*Double bond position indicated by a capital letter is unknown. DSummed features are groups of two or three fatty acids that could not be separated by the Microbial Identification System. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Optimal growth temperature is 25 uC; growth occurs at 15 and 35 uC, but not at 10 or 37 uC. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of 2.0 % (w/v) NaCl; growth occurs in the presence of 1.0–5.0 % (w/v) NaCl. Mg2+ ions are required for growth. H2S is not produced. Flexirubin-type pigments are not produced. Casein, starch and Tweens 20, 40, 60 and 80 are hydrolysed, but aesculin, CM-cellulose, gelatin, hypoxanthine, L-tyrosine, urea and xanthine are not. Acid is produced from Dgalactose, D-glucose, D-mannose, L-rhamnose, sucrose and D-xylose, but not from L-arabinose, cellobiose, D-fructose, lactose, maltose, melezitose, melibiose, raffinose, D-ribose, trehalose, myo-inositol, D-mannitol or D-sorbitol. Susceptible to ampicillin, carbenicillin, cephalothin, chloramphenicol, lincomycin, novobiocin, oleandomycin, penicillin G and tetracycline, but not to gentamicin, kanamycin, neomycin, polymyxin B or streptomycin. In the API ZYM system, alkaline phosphatase, leucine arylamidase and N-acetylb-glucosaminidase activities are present, but esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohdrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, amannosidase and a-fucosidase activities are absent. The type strain, AH-M5T (5KCTC 32578T5CECT 8487T), was isolated from a tidal flat sediment of Aphaedo, an island in the south-west sea, South Korea. The DNA G+C content of the type strain is 34.8 mol%.
ACKNOWLEDGEMENTS This work was supported by the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea Program for Collection, Management and Utilization of Biological Resources (grant NRF2013M3A9A5075953). We are grateful to the KACC for kindly providing Meridianimaribacter flavus KACC 15613T.
REFERENCES Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel’s
unidentified lipid. The DNA G+C content is 34.8 mol%. The type species is Seonamhaeicola aphaedonensis. A member of the family Flavobacteriaceae, phylum Bacteroidetes, according to 16S rRNA gene sequence analysis. Description of Seonamhaeicola aphaedonensis sp. nov. Seonamhaeicola aphaedonensis (ap.hae.do.nen9sis. N.L. masc. adj. aphaedonensis pertaining to Aphaedo, an island of South Korea, from where the type strain was isolated). Exhibits the following properties in addition to those given in the genus description. Cells are 0.2–0.7 mm in diameter and 0.9–8.5 mm in length. Colonies on MA agar are circular, slightly convex, smooth, glistening, strong orange–yellow in colour and 0.5–1.0 mm after incubation for 5 days at 25 uC. 1880
Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. 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, J. T. Staley, D. R. Brown, B. P. Hedlund, B. J. Paster, N. L. Ward, W. Ludwig & W. B. Whitman. 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. International Journal of Systematic and Evolutionary Microbiology 64
Seonamhaeicola aphaedonensis gen. nov., sp. nov. 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.
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.
Jeong, S. H., Park, M. S., Jin, H. M., Lee, K., Park, W. & Jeon, C. O. (2013). Aestuariibaculum suncheonense gen. nov., sp. nov., a marine
Park, S. C., Choe, H. N., Hwang, Y. M., Baik, K. S., Kim, S. N., Lee, Y. S., Jung, J. S. & Seong, C. N. (2013). Marinivirga aestuarii gen.
bacterium of the family Flavobacteriaceae isolated from a tidal flat and emended descriptions of the genera Gaetbulibacter and Tamlana. Int J Syst Evol Microbiol 63, 332–338.
nov., sp. nov., a member of the family Flavobacteriaceae, isolated from marine environments, and emended descriptions of the genera Hyunsoonleella, Jejuia and Pontirhabdus and the species Hyunsoonleella jejuensis, Jejuia pallidilutea and Pontirhabdus pectinivorans. Int J Syst Evol Microbiol 63, 1524–1531.
Jung, S.-Y., Kang, S.-J., Lee, M.-H., Lee, S.-Y., Oh, T.-K. & Yoon, J.-H. (2005). Gaetbulibacter saemankumensis gen. nov., sp. nov., a
novel member of the family Flavobacteriaceae isolated from a tidal flat sediment in Korea. Int J Syst Evol Microbiol 55, 1845–1849. Jung, Y.-T., Lee, J.-S. & Yoon, J.-H. (2013). Hwangdonia seohaensis
gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 3186–3191. Kim, J.-H., Kim, K.-Y., Hahm, Y.-T., Kim, B.-S., Chun, J. & Cha, C.-J. (2008). Actibacter sediminis gen. nov., sp. nov., a marine bacterium of
the family Flavobacteriaceae isolated from tidal flat sediment. Int J Syst Evol Microbiol 58, 139–143. Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in
bacterial systematics. Methods Microbiol 19, 161–207. Kwon, K. K., Lee, H.-S., Jung, H.-B., Kang, J.-H. & Kim, S.-J. (2006).
Yeosuana aromativorans gen. nov., sp. nov., a mesophilic marine bacterium belonging to the family Flavobacteriaceae, isolated from estuarine sediment of the South Sea, Korea. Int J Syst Evol Microbiol 56, 727–732. La´nyi, B. (1987). Classical and rapid identification methods for
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
2nd edn, vol. 4, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Sasser, M. (1990). Identification of bacteria by gas chromatography
of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128. Wang, B., Sun, F., Du, Y., Liu, X., Li, G., Lai, Q., Luo, J. & Shao, Z. (2010). Meridianimaribacter flavus gen. nov., sp. nov., a member of
the family Flavobacteriaceae isolated from marine sediment of the South China Sea. Int J Syst Evol Microbiol 60, 121–127. Yi, H. & Chun, J. (2011). Snuella lapsa gen. nov., sp. nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 61, 2469–2474. 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
medically important bacteria. Methods Microbiol 19, 1–67.
Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Lee, S.-Y., Lee, M.-H., Oh, T.-K. & Yoon, J.-H. (2012). Lutibacter
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
aestuarii sp. nov., isolated from a tidal flat sediment, and emended description of the genus Lutibacter Choi and Cho 2006. Int J Syst Evol Microbiol 62, 420–424. Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J Bacteriol 85, 1183–1184. Li, Y., Bai, S., Yang, C., Lai, Q., Zhang, H., Chen, Z., Wei, J., Zheng, W., Tian, Y. & Zheng, T. (2013). Mangrovimonas yunxiaonensis gen. nov.,
sp. nov., isolated from mangrove sediment. Int J Syst Evol Microbiol 63, 2043–2048. Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977).
Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol 27, 104–117.
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.
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454. Yoon, J.-H., Kang, S.-J., Jung, Y.-T. & Oh, T.-K. (2008). Aestuariicola saemankumensis gen. nov., sp. nov., a member of the family Flavobacteriaceae, isolated from tidal flat sediment. Int J Syst Evol Microbiol 58, 2126–2131. Yoon, J.-H., Lee, S.-Y. & Oh, T.-K. (2013). Gaetbulibacter lutimaris sp. nov., isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 995–1000.
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