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Ruegeria Conchae Sp. Nov., Isolated from the Ark Clam Scapharca Broughtonii

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Ruegeria Conchae Sp. Nov., Isolated from the Ark Clam Scapharca Broughtonii International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2851–2857 DOI 10.1099/ijs.0.037283-0 Ruegeria conchae sp. nov., isolated from the ark clam Scapharca broughtonii Jina Lee,13 Tae Woong Whon,13 Na-Ri Shin,1 Seong Woon Roh,1 Jandi Kim,1 Seong-Kyu Park,1 Min-Soo Kim,1 Kee-Sun Shin,2 Jung-Sook Lee,2 Keun Chul Lee,2 Young-Ok Kim3 and Jin-Woo Bae1 Correspondence 1Department of Life and Nanopharmaceutical Sciences and Department of Biology, Jin-Woo Bae Kyung Hee University, Seoul 130-701, Republic of Korea [email protected] 2Korean Collection for Type Cultures (KCTC), Biological Resource Center (BRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea 3Biotechnology Research Division, National Fisheries Research and Development Institute (NFRDI), Gijang, Busan 619-705, Republic of Korea A slightly halophilic, Gram-negative, strictly aerobic, non-motile rod, designated TW15T, was isolated from an ark clam in South Korea. Growth occurred at 10–37 6C, with 1–5 % (w/v) NaCl and at pH 7.0–10.0. Optimal growth occurred at 25–30 6C, with 2 % (w/v) NaCl and at pH 8.0. Strain TW15T exhibited both oxidase and catalase activities. The major fatty acids of strain TW15T were summed feature 8 (consisting of C18 : 1v7c and/or C18 : 1v6c) and 11-methyl C18 : 1v7c. The predominant isoprenoid quinone was ubiquinone-10 (Q-10). The polar lipids of strain TW15T comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and five unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TW15T was most closely related to Ruegeria lacuscaerulensis DSM 11314T (98.0 % 16S rRNA gene sequence similarity). DNA–DNA relatedness with closely related strains was ,52±3 %. The DNA G+C content was 55.7 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain TW15T represents a novel species of the genus Ruegeria, for which the name Ruegeria conchae sp. nov. is proposed. The type strain is TW15T (5KACC 15115T 5JCM 17315T). The genus Ruegeria was first introduced by Uchino et al. et al., 2007). R. pelagia (Lee et al., 2007) has been reported (1998) to accommodate Gram-negative, aerobic, oxidase- as a later heterotypic synonym of R. mobilis (Lai et al., and catalase-positive, non-phototrophic bacteria. At the 2010). All species of the genus Ruegeria have been isolated time of writing, eight species of the genus Ruegeria have from marine environments with the exception of R. been identified: Ruegeria lacuscaerulensis, R. pomeroyi lacuscaerulensis, which was isolated from a geothermal lake (Yi et al., 2007), R. mobilis (Muramatsu et al., 2007), (Petursdottir & Kristjansson, 1997). R. atlantica (Uchino et al., 1998), R. scottomollicae Strain TW15T was isolated from an ark clam (Scapharca (Vandecandelaere et al., 2008), R. marina (Huo et al., broughtonii) in the South Sea of Korea. Ark clams inhabit 2011), R. faecimaris (Oh et al., 2011) and R. halocynthiae mud flats from the intertidal zone to a depth of 50 m and (Kim et al., 2012). Ruegeria gelatinovorans and Ruegeria are farmed in large quantities for food. After being algicola have been reclassified as Thalassobius gelatinovorus harvested from an ark clam farm, one ark clam was (Arahal et al., 2005) and Marinovum algicola (Martens flash-frozen and then preserved at 280 uC until use. et al., 2006), respectively, and two species of the genus Homogenized tissue was suspended in sterilized PBS, Silicibacter, Silicibacter lacuscaerulensis and Silicibacter serially diluted and cultured on marine agar 2216 (MA; pomeroyi, have been transferred to the genus Ruegeria (Yi Difco) at 25 uC for 3 days. A pure culture was obtained after at least three subcultivations on MA and was stored as 3These authors contributed equally to this work. a suspension in 40 % glycerol at 280 uC. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TW15T is HQ171439. The 16S rRNA gene sequence of strain TW15T was Two supplementary figures are available with the online version of this amplified by colony PCR using PCR Pre-Mix (iNtRon paper. Biotechnology) with two universal bacterial primers: 037283 G 2012 IUMS Printed in Great Britain 2851 J. Lee and others forward primer 8F (59-AGAGTTTGATCCTGGCTCAG-39) analysis: R. lacuscaerulensis DSM 11314T, R. atlantica DSM and reverse primer 1492R (59-GGYTACCTTGTTACGA 5823T, R. pomeroyi DSM 15171T, R. faecimaris HD-28T, CTT-39). After purification using a QIAquick PCR obtained from the original isolator (Oh et al., 2011), Purification kit (Qiagen), sequencing was performed using R. marina JCM 16262T, R. mobilis DSM 23403T, R. a BigDye Terminator Cycle Sequencing Ready Reaction kit halocynthiae MA1-6T, obtained from the original isolator (Applied Biosystems), according to the manufacturer’s (Kim et al., 2012), and R. scottomollicae CCUG 55858T. instructions. To obtain a nearly full-length 16S rRNA gene Growth at 4, 10, 15, 25, 30, 37, 40, 45 and 55 uC was tested sequence, the sequence fragments were assembled in in triplicate in marine broth 2216 (MB; Difco). Growth SeqMan (DNASTAR). Comparison of the 16S rRNA gene with 0, 1, 2, 3, 4, 5, 8, 10, 12, 15 and 20 % (w/v) NaCl was sequence of strain TW15T with sequences in the EzTaxon determined in triplicate in MB that comprised all of the Server (Chun et al., 2007) clearly showed that the isolate constituents except NaCl and was supplemented with was most closely related to members of the genus Ruegeria appropriate concentrations of NaCl. Growth at pH 4.0– and exhibited 98.0 % 16S rRNA gene sequence similarity to 12.0 (in increments of one pH unit) was examined in R. lacuscaerulensis DSM 11314T, 97.8 % to R. atlantica triplicate at 30 uC in MB with the pH adjusted with the DSM 5823T, 97.5 % to R. halocynthiae MA1-6T, 96.9 % to following buffers: 10 mM MES (pH 4–6), 10 mM TAPS T T R. pomeroyi DSM 15171 , 96.2 % to R. faecimaris HD-28 , (pH 7–9) and 10 mM Na2HPO4 (pH 10–11). The condi- 95.7 % to R. mobilis DSM 23403T, 95.7 % to R. tions for growth of strain TW15T were 10–37 uC, 1–5 % scottomollicae CCUG 55858T and 95.0 % to R. marina (w/v) NaCl and pH 7.0–10.0 and for optimal growth were JCM 16262T. Multiple sequence alignment of the 16S 25–30 uC, 2 % (w/v) NaCl and pH 8.0. Based on the rRNA gene sequences of strain TW15T and members of requirement of NaCl for growth, strain TW15T was defined closely related species was carried out using CLUSTAL W as a slight halophile (Ventosa & Nieto, 1995). (Thompson et al., 1994) and the phylogenetic relationships Unless stated otherwise, all tests used to characterize were determined using MEGA5 (Tamura et al., 2011). The strain TW15T were performed under optimal conditions. neighbour-joining (Saitou & Nei, 1987), maximum- Morphological, physiological and biochemical analyses parsimony (Kluge & Farris, 1969) and maximum-like- were conducted after cultivation for 72 h at 30 uCinMB lihood (Felsenstein, 1981) methods were used to determine or on MA. Gram-staining was carried out using a Gram- phylogenetic distances from 1000, 1000 and 300 random staining kit (bioMe´rieux), according to the manufacturer’s bootstrap replicates, respectively. The results of the phylo- instructions, and cells were observed by light microscopy genetic analysis showed that strain TW15T belongs to a (Eclipse 50i; Nikon). Motility was examined according to cluster with other members of the genus Ruegeria (Fig. 1). the method of Tittsler & Sandholzer (1936) on semi-solid Based on the results of phylogenetic analysis, the following agar medium consisting of 0.3 % beef extract, 1 % strains were used as reference strains for comprehensive pancreatic digest of casein, 0.5 % sodium chloride and Ruegeria lacuscaerulensis DSM 11314T (ACNX01000031) 78/-/- Ruegeria atlantica DSM 5823T (D88526) Ruegeria conchae TW15T (HQ171439) 0.01 -/70/72 Ruegeria halocynthiae MA1-6T (HQ852038) DSM 15171T (CP000031) 75/-/- Ruegeria pomeroyi 97/90/94 Ruegeria marina JCM 16262T (FJ872535) Ruegeria faecimaris HD-28T (GU057915) Ruegeria mobilis DSM 23403T (AB255401) 95/77/85 Ruegeria scottomollicae CCUG 55858T (AM905330) Pseudoruegeria aquimaris SW-255T (DQ675021) 92/85/92 Pseudoruegeria lutimaris HD-43T (FJ374173) Thalassobius maritimus GSW-M6T (HM748766) T 92/-/82 Thalassobius aestuarii JC2049 (AY442178) 97/-/83 Thalassobius gelatinovorus IAM 12617T (D88523) -/72/- Thalassobius mediterraneus XSM19T (AJ878874) Stappia stellulata IAM 12621T (D88525) Fig. 1. Consensus 16S rRNA gene sequence phylogenetic tree constructed using the neighbour-joining, maximum-parsimony and maximum-likelihood algorithms, showing the position of strain TW15T in the genus Ruegeria. Bootstrap values (¢70 %) based on 1000, 1000 and 300 replicates for the neighbour-joining, maximum-parsimony and maximum-likelihood algorithms, respectively, are shown at branch nodes. Filled diamonds indicate that the corresponding nodes were recovered with all three algorithms. Stappia stellulata IAM 12621T was used as an outgroup. Bar, 0.01 substitutions per nucleotide. 2852 International Journal of Systematic and Evolutionary Microbiology 62 Ruegeria conchae sp. nov. 0.4 % agar. Cell morphology was observed by light R. atlantica DSM 5823T were cultured at 30 uC on MA for microscopy and energy-filtering transmission electron 3 days. The physiological ages of strain TW15T and the microscopy (Libra 120; Carl Zeiss) (Fig. S1, available in reference strains were standardized to the exponential IJSEM Online). For transmission electron micrography, a phase. The cellular fatty acids were saponified, methylated single colony of the isolate was suspended in 500 ml sterile and extracted according to the protocol of the Sherlock PBS buffer. Copper grids with 200-mesh carbon-coated Microbial Identification System (MIDI, 1999). The ex- Formvar were floated on a droplet of the suspension, tracted cellular fatty acids were analysed by GC (Hewlett negatively stained with 2 % uranyl acetate for 10 s, washed Packard 6890) and identified using the Microbial Iden- with deionized water two or three times and air-dried.
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