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Roseivivax Atlanticus Sp. Nov., Isolated from Surface Seawater of the Atlantic Ocean

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Roseivivax Atlanticus Sp. Nov., Isolated from Surface Seawater of the Atlantic Ocean Roseivivax atlanticus sp. nov., isolated from surface seawater of the Atlantic Ocean Guizhen Li, Qiliang Lai, Xiupian Liu, Fengqin Sun & Zongze Shao Antonie van Leeuwenhoek Journal of Microbiology ISSN 0003-6072 Volume 105 Number 5 Antonie van Leeuwenhoek (2014) 105:863-869 DOI 10.1007/s10482-014-0140-5 1 23 Your article is protected by copyright and all rights are held exclusively by Springer International Publishing Switzerland. This e- offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. 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The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Antonie van Leeuwenhoek (2014) 105:863–869 DOI 10.1007/s10482-014-0140-5 ORIGINAL PAPER Roseivivax atlanticus sp. nov., isolated from surface seawater of the Atlantic Ocean Guizhen Li • Qiliang Lai • Xiupian Liu • Fengqin Sun • Zongze Shao Received: 19 December 2013 / Accepted: 11 February 2014 / Published online: 25 February 2014 Ó Springer International Publishing Switzerland 2014 Abstract A taxonomic study was carried out on strain isoporae LMG 25204T (97.0 %); other species of 22II-S10sT, which was isolated from the surface seawa- genus Roseivivax shared 95.2–96.7 % sequence terof the Atlantic Ocean. The bacterium was found to similarity. The DNA–DNA hybridization estimate be Gram-negative, oxidase and catalase positive, rod values between strain 22II-S10sT and the two type shaped and motile by subpolar flagella. The isolate was strains (R. halodurans JCM 10272T and R. isoporae capable of gelatine hydrolysis but unable to reduce LMG 25204T) were 22.00 and 21.40 %. The principal nitrate to nitrite or degrade Tween 80 or aesculin. Growth fattyacidswereidentifiedasSummedFeature8 was observed at salinities of 0.5–18 % (optimum, (C18:1x7c/x6c) (67.4 %), C18:0 (7.2 %), C19:0 cyclo 2–12 %), at pH of 3–10 (optimum, 7) and at tempera- x8c (7.1 %), C18:1x7c 11-methyl (6.8 %) and C16:0 tures of 10–41 °C(optimum28°C). Phylogenetic (5.9 %). The respiratory quinone was determined to be analysis based on 16S rRNA gene sequences indicated Q-10 (100 %). Phosphatidylcholine, phosphatidyletha- that strain 22II-S10sT belongs to the genus Roseivivax, nolamine, phosphatidylglycerol, an aminolipid, a glyco- with highest sequence similarity to Roseivivax halodu- lipid and three phospholipids were present. The G?C rans JCM 10272T (97.2 %), followed by Roseivivax content of the chromosomal DNA was determined to be 67.5 mol%. The combined genotypic and phenotypic data show that strain 22II-S10sT represents a novel species within the genus Roseivivax, for which the name Rosei- Guizhen Li and Qiliang Lai contributed equally to this work. vivax atlanticus sp. nov. is proposed, with the type strain T T T Electronic supplementary material The online version of 22II-S10s (= MCCC 1A09150 = LMG 27156 ). this article (doi:10.1007/s10482-014-0140-5) contains supple- mentary material, which is available to authorized users. Keywords Roseivivax atlanticus sp. nov. Á G. Li Á Q. Lai Á X. Liu Á F. Sun Á Z. Shao (&) Atlantic Ocean Á Surface seawater Á DDH Á State Key Laboratory Breeding Base of Marine Genetic Taxonomy Resources, Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Key Laboratory of Abbreviation Marine Genetic Resources of Fujian Province, Collaborative Innovation Center of Deep Sea Biology, MCCC Marine Culture Collection of China Xiamen 361005, China e-mail: [email protected] Introduction G. Li Á Q. Lai Life Science College, Xiamen University, During an investigation of bacterial diversity in Xiamen 361005, China the surface seawater of the Atlantic Ocean, strain 123 Author's personal copy 864 Antonie van Leeuwenhoek (2014) 105:863–869 22II-S10sT was isolated and characterized taxo- N,N,N0,N0-tetramethyl-1,4-phenylenediamine. Catalase nomically along with many other bacterial isolates. activity was determined by bubble production in 10 % Comparative 16S rRNA gene sequence analysis (v/v) H2O2 solution. Cell motility was observed by the indicated that strain 22II-S10sT belongs to the genus hanging-drop method (Skerman 1967). The growth Roseivivax. The genus Roseivivax was proposed by temperature, pH range and tolerance to NaCl were tested Suzuki and colleagues (Suzuki et al. 1999) and according to the methods described (Li et al. 2013). presently comprises six species: Roseivivax halodu- Hydrolysis of starch, dried skimmed milk, cellulose and rans and Roseivivax halotolerans (Suzuki et al. Tweens20,40,60and80weretestedonmarineagar 1999), Roseivivax lentus (Park et al. 2010), Rosei- 2216 plates supplemented with 0.2 % (w/v) soluble vivax isoporae (Chen et al. 2012), Roseivivax starch, 0.5 % dried skimmed milk, 0.8 % cellulose or sediminis (Xiao et al. 2012) and Roseivivax pacificus 0.5 % (v/v) Tweens 20, 40, 60 or 80, 0.2 % DNA (Wu et al. 2013). Consequently, the aim of the according to standard methods (Dong and Cai 2001). present work was to determine the exact taxonomic Antibiotic susceptibility tests were performed using the position of strain 22II-S10sT by using a polyphasic disc diffusion method as described previously (Shieh approach. et al. 2003). API 20NE, API 20E, and API ZYM strips (bioMe´rieux) were carried out according to the manu- facturer’s instructions, with the single modification of Materials and methods adjusting the NaCl concentration to 3.0 % in all tests. Bacterial strains, isolation, and cultivation Determination of 16S rRNA gene sequence and phylogenetic analysis Surface seawater was sampled at site (W14.4°, S14.1°) in 2011 during cruise DY-115A of the South Atlantic Genomic DNA was extracted using a kit (SBS Ocean on the R/V ‘‘Da-Yang Yi-Hao’’. The surface Genetech Co., Ltd. China) and the 16S rRNA gene seawater was diluted and spread on marine agar 2216 was amplified by PCR using primers described medium (BD Difco). After one month of aerobic previously (Liu and Shao 2005). Sequence similarity incubation at 25 °C, the colonies were picked. Purity was determined using the EzTaxon-e server (Kim was confirmed by the uniformity of cell morphology et al. 2012). Sequences of related taxa were obtained after repeated restreaking. For morphological and from the GenBank database. Phylogenetic analysis biochemical characterization, strain 22II-S10sT was was performed using MEGA version 5.0 (Tamura cultivated on marine agar 2216 medium (BD Difco) et al. 2011) with distance options according to the unless otherwise indicated. R. halotolerans JCM Kimura two-parameter model and clustering with 10271T, R. halodurans JCM 10272T and R. isoporae the neighbour-joining (Saitou and Nei 1987), min- LMG 25204T were obtained from JCM (Japan Collec- imum evolution (Rzhetsky and Nei 1992), and tion of Microorganisms) and LMG (Laboratorium maximum likelihood (Felsenstein 1981) methods, Microbiologie, Universiteit Gent, Belgium) as refer- and supported with bootstrap values based on 1,000 ence strains and cultured under the same conditions for replications. comparative testing. Genome sequencing and DNA–DNA Phenotypic characterization hybridization (DDH) estimation Gram staining was performed using a Gram staining kit The genome sequences strain 22II-S10sT, R. halodu- (Hangzhou Tianhe Microorganism Reagent Co.) accord- rans JCM 10272T and R. isoporae LMG 25204T were ing to the manufacturer’s instructions. The cell mor- determined by Shanghai Majorbio Bio-pharm Tech- phology, size, and flagellation pattern were observed by nology Co., Ltd. (Shanghai, China), using Solexa transmission electron microscopy (JEM-1230, JEOL) of paired-end (500 bp library) sequencing technology. cells negatively stained with phosphotungstic acid after The G?C content of the chromosomal DNA of strain culture on marine agar at 28 °C for 1 day. Oxidase 22II-S10sT was determined according to draft genome activity was evaluated by the oxidation of 1 % (w/v) sequence. 123 Author's personal copy Antonie van Leeuwenhoek (2014) 105:863–869 865 DNA–DNA hybridization (DDH) estimate values 16S rRNA gene sequence analysis among them were analyzed using the genome-to- genome distance calculator (GGDC2.0) (Auch et al. A nearly full-length 16S rRNA gene sequence (1,426 2010a, b; Meier-Kolthoff et al. 2013). nt, GenBank accession number KF906718.) from strain 22II-S10sT was determined. The 22II-S10sT Determination of fatty acid, isoprenoid 16S rRNA gene showed the highest sequence simi- ubiquinone, and polar lipid compositions larity to R. halodurans JCM 10272T (97.2 %), followed by R. isoporae LMG 25204T (97.0 %); other Fatty acids in whole cells grown on marine agar 2216 species of the genus Roseivivax shared 95.2–96.7 % medium at 28 °C for 48 h were saponified, methyl- sequence similarity. As shown in Fig. 1, a phyloge- ated, and extracted using the standard MIDI (Sherlock netic tree was constructed based on the 16S rRNA Microbial Identification System, version 6.0B) proto- gene sequences of all six species of the genus col. Three reference type strains (R. halodurans JCM Roseivivax and representatives of related genera of 10272T, R. isoporae LMG 25204T and R. halotolerans the family Rhodobacteraceae. This analysis showed JCM 10271T) were tested at the same time under the that strain 22II-S10sT formed a clade with R. halodu- same conditions. The fatty acids were analysed by gas rans JCM 10272T and R. isoporae LMG 25204T chromatography (Agilent Technologies 6850) and within the genus Roseivivax. This topology was identified using the TSBA6.0 database of the Micro- confirmed by the maximum-likelihood and minimum bial Identification System (Sasser 1990).
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