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Halomonas Marisflavae Sp. Nov., a Halophilic Bacterium Isolated from the Yellow Sea in Korea

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Halomonas Marisflavae Sp. Nov., a Halophilic Bacterium Isolated from the Yellow Sea in Korea International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1171–1177 Printed in Great Britain Halomonas marisflavae sp. nov., a halophilic bacterium isolated from the Yellow Sea in Korea 1 Korea Research Institute of Jung-Hoon Yoon,1 Seong Hyun Choi,3 Keun-Chul Lee,1 Yung Hee Kho,1 Bioscience and 4 1,2 Biotechnology (KRIBB), Kook Hee Kang and Yong-Ha Park PO Box 115, Yusong, Taejon, Korea Author for correspondence: Yong-Ha Park. Tel: 82 42 860 4620. Fax: 82 42 860 4598. 2 j j Probionic Corporation. Bio- e-mail: yhpark!mail.kribb.re.kr venture Centre, Korea, Research Institute of Bioscience and T Biotechnology (KRIBB), A halophilic Gram-negative bacterial strain, SW32 , which was isolated from a PO Box 115, Yusong, sample from the Yellow Sea of Korea, was subjected to a polyphasic taxonomic Taejon, Korea study. This organism grew optimally in the presence of 05–12% NaCl. On the 3 Department of Food basis of phenotypic and phylogenetic data, strain SW32T appeared to be a Science and Technology, member of the genus Halomonas. Strain SW32T formed a distinct evolutionary Chungnam National University, Taejon, Korea lineage within the phylogenetic clade comprising Halomonas species and the genera Zymobacter and Carnimonas. The 16S rDNA sequence of strain SW32T 4 Department of Food and Life Science, contains 19 signature characteristics of the genus Halomonas and the family Sungkyunkwan University, Halomonadaceae. Strain SW32T possessed a single polar flagellum, ubiquinone- Chunchun-dong 300, 9 as the predominant respiratory lipoquinone and C ,C and C ω7c Jangan-gu, Suwon, Korea 18:1 16:0 16:1 and/or iso-C15:0 2OH as the major fatty acids. The DNA GMC content was 59 mol%. Levels of 16S rDNA similarity between strain SW32T and the type strains of all validly described Halomonas species were 920–938%. Strain SW32T exhibited 16S rDNA similarity values of 927% to Zymobacter palmae IAM 14233T and 916% to Carnimonas nigrificans CECT 4437T. These data indicate that strain SW32T was related enough to members of the genus Halomonas to be placed as a new species within that genus. Therefore the name Halomonas marisflavae sp. nov. is proposed for strain SW32T. The type strain of the new species is strain SW32T (l KCCM 80003T l JCM 10873T). Keywords: Halomonas marisflavae sp. nov., polyphasic taxonomy, marine bacterium INTRODUCTION eurihalina is closely related to Halomonas elongata, the type species of the genus Halomonas (Mellado et al., The family Halomonadaceae was originally created by 1995). Accordingly, the genera Halomonas, Deleya Franzmann et al. (1989) to accommodate the genera and Halovibrio and the species Paracoccus halo- Halomonas (Vreeland et al., 1980) and Deleya denitrificans were recently unified into a single genus (Baumann et al., 1983). However, Halomonas and Halomonas (Dobson & Franzmann, 1996) and Deleya, together with the genus Halovibrio (Fendrich, Volcaniella eurihalina was transferred to the genus 1988), lacked differential phenotypic, particularly Halomonas as Halomonas eurihalina (Mellado et al., chemotaxonomic, characters (Franzmann & Tindall, 1995). 1990; Skerratt et al., 1991). Phylogenetic analyses based on 16S rRNA gene sequences showed that the The genus Zymobacter, which was proposed by members belonging to the three genera form a mono- Okamoto et al. (1993), has been included in the family phyletic group (Dobson et al., 1993) and Volcaniella Halomonadaceae (Dobson & Franzmann, 1996). The genus Chromohalobacter, with only a single species, ................................................................................................................................................. Chromohalobacter marismortui (Ventosa et al., 1989), Abbreviations: FAME, fatty acid methyl ester; MA, marine agar; TSA(B), falls within the phylogenetic radiation occupied by trypticase soy agar (broth). Halomonas species, although Mellado et al. (1995) The GenBank accession number for the 16S rDNA sequence of strain SW32T concluded that there are enough phenotypic is AF251143. differences to warrant classification of Chromo- 01631 # 2001 IUMS 1171 J.-H. Yoon and others halobacter as a genus separate from the Halo- was determined using the bioMe! rieux Gram Strain kit monas\Deleya complex. A new genus, Carnimonas, according to the manufacturer’s instructions. Catalase ac- which contains only a single species, Carnimonas tivity was determined by bubble formation in a 3% hydrogen nigrificans, and forms a monophyletic group with peroxide solution. Oxidase activity was determined by Zymobacter palmae, has been described (Garriga et al., oxidation of 1% tetramethyl-p-phenylenediamine. Nitrate reduction and hydrolysis of aesculin were determined as 1998). Despite its chemotaxonomic and physiological described by Lanyi (1987). Hydrolysis of casein, gelatin, similarities, the authors did not include the genus hypoxanthine, starch, Tween 80, tyrosine and xanthine, and Carnimonas within the family Halomonadaceae be- production of urease were determined as described by cause it lacked 2 out of 15 descriptive signature Cowan & Steel (1965). Acid production from carbohydrates characteristics in its 16S rRNA sequence (Garriga was determined by using the API 50CH system (bio- et al., 1998). Thus, the family Halomonadaceae cur- Me! rieux). Other physiological tests were performed with rently possesses three genera; Halomonas (Vreeland the API 20NE system (bioMe! rieux). Growth under an- et al., 1980; Dobson & Franzmann, 1996), Chromo- aerobic conditions was determined after incubation in an halobacter (Ventosa et al., 1989) and Zymobacter anaerobic chamber with MA that was prepared anaero- (Okamoto et al., 1993). bically. Tolerance of NaCl was measured in TSB containing no NaCl or 0n5–30% (w\v) NaCl. Growth at various Members of the family Halomonadaceae are Gram- temperatures was measured on MA. negative, either slight or moderate halophiles with Chemotaxonomic characterization. The presence or absence straight or curved, rod-shaped cells. The family Halo- of diaminopimelic acid in the peptidoglycan was determined monadaceae is characterized by having ubiquinone-9 as described by Komagata & Suzuki (1987). Respiratory as the predominant respiratory lipoquinone and C"):" lipoquinones were extracted and purified as described by plus cyclo C"*:! and C"':! as the major fatty acids. This Komagata & Suzuki (1987). The purified ubiquinones were family is a member of the γ-subclass of the class dissolved in acetone and separated by reverse-phase HPLC. Proteobacteria. For quantitative analysis of cellular fatty acid compositions, a loop of cell mass was harvested and FAMEs were prepared Recently, a halophilic Gram-negative bacterial or- and identified following the instructions of the Microbial ganism, strain SW32, was isolated from a water sample Identification System (MIDI). of the Yellow Sea, Korea. The 16S rDNA sequence analysis indicated that this organism lies at the Determination of GjC content. Chromosomal DNA was isolated and purified according to the method described periphery of the phyletic radiation encompassed by the previously (Yoon et al., 1996) with the exception that genus Halomonas and is phylogenetically related to the ribonuclease T1 was used together with ribonuclease A. The genera Zymobacter and Carnimonas. This observation GjC content was determined by the method of Tamaoka & suggested that other useful taxonomic data were also T Komagata (1984). DNA was hydrolysed and the resultant required for reliable classification of strain SW32 . nucleotides were analysed by reverse-phase HPLC. Accordingly, the aim of the present study was to T 16S rDNA sequencing and phylogenetic analysis. 16S rDNA unravel the taxonomic status of strain SW32 with a was amplified by PCR using the two universal primers as combination of phenotypic characters and detailed described previously (Yoon et al., 1998). The PCR product phylogenetic analysis. was purified with a QIAquick PCR purification kit (Qiagen) and the 16S rDNA was sequenced using the ABI PRISM METHODS BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The purified sequencing reaction Bacterial strain and cultural conditions. Strain SW32T was mixtures were automatically electrophoresed using an Ap- isolated from a water sample of the Yellow Sea in Boryung plied Biosystems model 310 automatic DNA sequencer. City, Korea, by the dilution plating technique on marine Alignment of sequences was carried out with agar (MA) (Difco). For the investigation of morphological software (Thompson et al., 1994). Gaps at the 5h and 3h ends and physiological characteristics, strain SW32T was cul- of the alignment were omitted from further analyses. tivated on MA and broth (Difco) and trypticase soy agar Phylogenetic trees were inferred by using three tree-making (TSA) and broth (TSB) (BBL) at 28 mC. Cell biomass for algorithms, neighbour-joining (Saitou & Nei, 1987), respiratory lipoquinone analysis and DNA extraction was maximum-likelihood (Felsenstein, 1981) and maximum- obtained from a marine broth culture. Strain SW32T was parsimony (Kluge & Farris, 1969) methods in the cultivated at 28 mC on a horizontal shaker at 150 r.p.m. and package (Felsenstein, 1993). Evolutionary distance matrices the broth culture was checked for purity microscopically for the neighbour-joining method were calculated with the before being harvested by centrifugation. For fatty acid algorithm of Jukes & Cantor (1969) with the methyl ester (FAME) analysis, cell mass of strain SW32T program. The stability
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