International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4689–4694 DOI 10.1099/ijsem.0.000635 Simplicispira piscis sp. nov., isolated from the gut of a Korean rockfish, Sebastes schlegelii Dong-Wook Hyun, Sei Joon Oh, Min-Soo Kim, Tae Woong Whon, Mi-Ja Jung, Na-Ri Shin, Pil Soo Kim, Hyun Sik Kim, June-Young Lee, Woorim Kang and Jin-Woo Bae Correspondence Department of Life and Nanopharmaceutical Sciences and Department of Biology, Jin-Woo Bae Kyung Hee University, Seoul 130-701, Republic of Korea [email protected] A novel Gram-stain-negative, aerobic, motile and rod-shaped bacterium, designated strain RSG39T, was isolated from the gut of a Korean rockfish, Sebastes schlegelii. The 16S rRNA gene sequence analysis revealed that strain RSG39T belonged to the genus Simplicispira in the class Betaproteobacteria and its highest sequence similarity was shared with S. psychrophila (98.4 %). The isolate grew optimally at 20 8C, at pH 7 and with 0 % (w/v) NaCl. The main respiratory quinone of the isolate was ubiquinone Q-8. The major cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and summed feature 8 (C18 : 1v7c and/or C18 : 1v6c). The polar lipids of the isolate were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and six unidentified lipids. The DNA–DNA hybridization values showed ,7.4 % genomic relatedness with closely related strains. The genomic DNA G+C content was 65.2 mol %. Based on phylogenetic, phenotypic, chemotaxonomic and genotypic analyses, strain RSG39T represents a novel species of the genus Simplicispira, for which the name Simplocospira piscis sp. nov. is proposed. The type strain is RSG39T (5KACC 17539T5JCM 19291T). The genus Simplicispira belongs to the family Comamona- abilities (Lee et al., 2000). Commensal or foreign microbes, daceae within the class Betaproteobacteria, and was pro- which have probiotic or pathogenic effects on hosts, affect posed by Grabovich et al. (2006). The members of the aquaculture efficiency and determine success or failure of genus Simplicispira are defined as Gram-staining-negative, aquaculture (Korkea-aho et al., 2011; Santander et al., aerobic, rod-shaped bacteria that contain ubiquinone-8 2013; Vine et al., 2004). Due to the importance of gut com- (Q-8) as a major quinone. At the time of writing, the mensal microbiota on the fishery industry, we have inves- genus Simplicispira comprises three species with validly tigated microbiota of marine species and reported novel published names: Simplicispira metamorpha, Simplicispira intestinal bacteria (Hyun et al., 2013, 2014). During this psychrophila (Grabovich et al., 2006) and Simplicispira ongoing study, we isolated a novel bacterial strain, desig- limi (Lu et al., 2007). nated strain RSG39T, from Sebastes schlegelii. This paper describes the taxonomic characterization of this novel bac- The Korean rockfish Sebastes schlegelii is recognized as a terial isolate based on a polyphasic analysis, which suggests commercially important ovoviviparous marine species in that the isolate represents a novel species. Korea and is distributed throughout shallow rocky shores along the coasts of Korea, Japan and China. Aquaculture To isolate intestinal bacteria, the detached and homogen- of Sebastes schlegelii has been widely developed in Korea ized intestinal tissue of farmed Korean rockfish Sebastes due to several beneficial characteristics for farming such schlegelii was inoculated onto R2A agar (Difco) plates as viviparous reproduction and environmental adaptation using the dilution-plating technique and incubated at 2 20 8C. Strain RSG39T was isolated from a 10 2-diluted sample after cultivation at 20 8C for 72 h. The isolate was 2 8 Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanola- purified by repeated subculture and stored at 80 Casa mine; PG, phosphatidylglycerol. suspension in R2A broth (MBcell) containing 40 % (v/v) glycerol. All physiological, biochemical, chemotaxonomic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RSG39T is KC884002. and genotypic analyses were repeated at least three times. Four supplementary figures and a supplementary table are available with A phylogenetic analysis was performed based on 16S rRNA the online Supplementary Material. gene sequences. The 16S rRNA gene sequence of the isolate Downloaded from www.microbiologyresearch.org by 000635 G 2015 IUMS Printed in Great Britain 4689 IP: 163.180.66.204 On: Tue, 01 Mar 2016 10:41:03 D.-W. Hyun and others was amplified using Ex Taq PCR premix (Takara) with the sequences were aligned using CLUSTAL W (Thompson universal bacterial primer pair, forward primer 8F and et al., 1994) and used to reconstruct phylogenetic trees reverse primer 1492R (Lane, 1991). The 16S rRNA gene with MEGA 6(Tamuraet al., 2013). The phylogenetic dis- amplicon was sequenced using a BigDye Terminator tances were calculated using the neighbour-joining (Saitou Cycle Sequencing Ready Reaction kit (Applied Biosystems), & Nei, 1987), maximum-parsimony (Fitch, 1972) and according to the manufacturer’s instructions. The reaction maximum-likelihood (Felsenstein, 1981) algorithms based mixtures were analysed using an automated DNA analyser on 1000 bootstrap replicates. The phylogenetic trees (3730xl DNA Analyser; Applied Biosystems). The sequenced showed that strain RSG39T formed a cluster with members 16S rRNA gene sequence fragments were assembled using of the genus Simplicispira (Fig. 1 and Figs S1-2, available in SeqMan 5.0 (DNASTAR) and compared with the sequences the online Supplementary Material). To facilitate a more of type strains in the EzTaxon-e server (http://eztaxon-e. comprehensive and comparative characterization of ezbiocloud.net/; Kim et al., 2012). The 16S rRNA gene strain RSG39T, type strains of all species of the genus Sim- sequences comparison revealed that strain RSG39T shared plicispira were used as reference strains. S. psychrophila LMG 98.4 % similarity with S. psychrophila LMG 5408T,97.9% 5408T and S. metamorpha LMG 23381T were obtained from similarity with S. limi KCTC 12608T and 97.3 % similarity the Belgian Co-ordinated Collections of Microorganisms/ with S. metamorpha LMG 23381T. A phylogenetic consensus Laboratorium voor Microbiologie, Universiteit Gent tree was reconstructed to clarify the phylogenetic relation- (BCCM/LMG), and S. limi KCTC 12608T was obtained ships between the isolate and closely related species. The from the Korean Collection for Type Cultures (KCTC). Acidovorax defluvii BSB411T (Y18616) Acidovorax radicis N35T (AFBG01000030) 0.01 CCUG 11779T (AF078766) 72/-/76 Acidovorax temperans Acidovorax facilis CCUG 2113T (AF078765) 96/93/96 Acidovorax soli BL21T (FJ599672) Acidovorax wautersii NF 1078T (JQ946365) Alicycliphilus denitrificans K601T (CP002657) Diaphorobacter aerolatus 8604S-37T (KC352658) Diaphorobacter oryzae RF3T (EU342381) 99/-/74 Diaphorobacter nitroreducens NA10BT (AB064317) 89/-/82 Xenophilus aerolatus 5516S-2T (EF660342) 82/-/76 Xenophilus arseniciresistens YW8T (KC010298) Xylophilus ampelinus ATCC 33914T (AF078758) 81/-/74 Simplicispira metamorpha LMG 23381T (Y18618) T KCTC 12608 (DQ372987) 86/88/88 Simplicispira limi T 97/94/97 Simplicispira piscis RSG39 (KC884002) 88/91/94 Simplicispira psychrophila LMG 5408T (JHYS01000032) Comamonas granuli Ko03T (AB187586) Delftia lacustris DSM 21246T (EU888308) Burkholderia cepacia ATCC 25416T (AXB001000009) Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain RSG39T and closely related species. The tree was reconstructed using the neighbour-joining (NJ) algorithm. Filled diamonds represent identical branches generated using three algorithms: NJ, maximum-parsimony (MP) and maximum-likelihood (ML). MP-and ML-based phylogenetic trees are presented in Figs S1 and S2, respectively. Numbers at nodes indicate bootstrap values (NJ/MP/ML) as percentages of 1000 replicates; values ,70 % are not shown. Burkholderia cepacia ATCC 25416T (GenBank accession no. AXBO01000009) was used as an outgroup. Bar, 0.01 accumulated changes per nucleotide. Downloaded from www.microbiologyresearch.org by 4690 International Journal of Systematic and Evolutionary Microbiology 65 IP: 163.180.66.204 On: Tue, 01 Mar 2016 10:41:03 Simplicispira piscis sp. nov. The following characteristics were determined using cells activities and acid production from various carbohydrates of strain RSG39T grown on R2A at 20 8C for 48 h. (Table 1). The results of the biochemical tests for the isolate Gram-staining was performed using a Gram staining kit are detailed in the species description. (bioMe´rieux) according to the manufacturer’s instructions. To determine the chemotaxonomic characteristics of strain Gram-staining and cell morphology were observed using a RSG39T, the cellular fatty acid profile, isoprenoid quinone light microscope (Eclipse 50i; Nikon). Cell motility was composition and polar lipid composition were compared tested in semi-solid R2A broth containing 0.4 % agar with those of the reference strains. Chemotaxonomic ana- (Tittsler & Sandholzer, 1936). A transmission electron lyses were performed using cell masses of the isolate and microscope (SUPRA VP55; Zeiss) was used to detect the T the reference strains after cultivation on R2A in the opti- presence of flagella. Cells of strain RSG39 were Gram- mum growth conditions for 48 h. Cellular fatty acids m stain-negative, rod-shaped (0.7–0.9 m wide and 1.0– were saponified, methylated and extracted, according to 2.8