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Jeotgalibacillus Alimentarius Gen. Nov., Sp. Nov., a Novel Bacterium

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Jeotgalibacillus Alimentarius Gen. Nov., Sp. Nov., a Novel Bacterium International Journal of Systematic and Evolutionary Microbiology (2001), 51, 2087–2093 Printed in Great Britain Jeotgalibacillus alimentarius gen. nov., sp. nov., a novel bacterium isolated from jeotgal with L-lysine in the cell wall, and $ reclassification of Bacillus marinus Ruger 1983 as Marinibacillus marinus gen. nov., comb. nov. 1 Korea Research Institute of Jung-Hoon Yoon,1 Norbert Weiss,4 Keun-Chul Lee,1 In-Sun Lee,2 Bioscience and 3 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 42 862 1315. 2 j j Probionic Corporation, e-mail: yhpark!mail.kribb.re.kr Bio-venture Centre, Korea Research Institute of Bioscience and T Biotechnology (KRIBB), A moderately halophilic, round-endospore-forming bacterium (strain YKJ-13 ) PO Box 115, Yusong, was isolated from jeotgal, a traditional Korean fermented seafood, and Taejon, Korea studied by a polyphasic taxonomic approach. This organism was related to the 3 Department of Food and phylogenetic clade comprising members of Bacillus rRNA group 2 and formed a Life Science, cluster with Bacillus marinus with a bootstrap fidelity value of 936%. The Sungkyunkwan University, Chunchun-dong 300, peptidoglycan type was A1α linked directly through L-Lys. Based on cell Jangan-gu, Suwon, Korea morphology, peptidoglycan type and phylogeny, strain YKJ-13T, together with 4 DSMZ – Deutsche B. marinus, is considered to be a member of Bacillus rRNA group 2. Strain YKJ- Sammlung von 13T was also characterized by having MK-7 and MK-8 as the predominant Mikroorganismen und menaquinones and iso-C15:0 as the major fatty acid. The DNA GMC content was Zellkulturen GmbH, T Mascheroder Weg 1b, 44 mol%. Strain YKJ-13 exhibited a 16S rDNA similarity value of 957% with B. D-38124 Braunschweig, marinus DSM 1297T, its closest phylogenetic relative. Levels of 16S rDNA Germany similarity between strain YKJ-13T and other Bacillus spp. were less than 942%. Therefore, on the basis of the data presented, the name Jeotgalibacillus alimentarius gen. nov., sp. nov. is proposed for strain YKJ-13T (l KCCM 80002T l JCM 10872T). It is also proposed that B. marinus be reclassified in Marinibacillus gen. nov. as Marinibacillus marinus comb. nov. Keywords: Jeotgalibacillus alimentarius, Bacillus marinus, Marinibacillus marinus, Bacillus rRNA group 2, jeotgal INTRODUCTION moderately halophilic bacterium (strain YKJ-13T)was studied in this report. Jeotgal, one of the representative traditional foods of Formation of round endospores is a typical charac- Korea, is prepared through blending of various kinds teristic of organisms assigned to Bacillus rRNA group of seafoods, including seawater and other ingredients, 2, which are characterized by having -lysine or - and becomes palatable through proper preservation. ornithine at position 3 of the peptide subunit of the Preliminary studies on the microbiology of jeotgal peptidoglycan (Rheims et al., 1999; Stackebrandt et have demonstrated the presence of a variety of al., 1987). Bacillus species belonging to this group form bacterial strains; the majority of the isolates were the radiation of a cluster distinct from members of Gram-positive or -variable, endospore-forming bacilli. other rRNA groups of the genus Bacillus (Ash et al., Considering their halotolerant or halophilic physio- 1991; Stackebrandt et al., 1987). However, it should be logical properties, most of bacterial isolates appear to noted that this group also consists of a phylogenetic have originated from seafoods and seawater. Among jumble with some non-Bacillus-type organisms, such many bacilli from jeotgal, a round-endospore-forming, as the genera Caryophanon, Planococcus, Filibacter ................................................................................................................................................. and Sporosarcina (Clausen et al., 1985; Farrow et al., The GenBank accession number for the 16S rDNA sequence of strain 1992, 1994; Stackebrandt et al., 1987). On the basis of YKJ-13T is AF281158. these properties, it has been recognized that the - 01860 # 2001 IUMS 2087 J.-H. Yoon and others lysine- and -ornithine-containing bacilli belonging to after air-drying, the grids were examined with a model CM- rRNA group 2 may have to be taxonomically re- 20 transmission electron microscope (Philips). evaluated (Farrow et al., 1994; Rheims et al., 1999). Physiological characterization. All physiological tests were In the present work, the morphological, phenotypic performed at 30 mC, except for the study of temperature T range for growth. Oxidase activity was determined by and phylogenetic characteristics of strain YKJ-13 are examining oxidation of 1% p-aminodimethylaniline oxalate. described in order to unravel its exact taxonomic Catalase activity was determined by bubble formation in a status. This organism has taxonomic properties in- 3%(v\v) hydrogen peroxide solution. Hydrolysis of aesculin dicative of rRNA group 2 bacilli. Results of 16S rDNA and nitrate reduction were determined as described pre- T sequence comparison showed that strain YKJ-13 viously (Lanyi, 1987). Hydrolyses of casein, gelatin, starch is phylogenetically most closely related to Bacillus and Tween 80, and urease activity were determined as marinus, with formation of a coherent cluster. B. described by Cowan & Steel (1965). Hydrolyses of hypox- marinus was later elevated to species rank by Ru$ ger anthine, tyrosine and xanthine were performed on marine (1983) with strains that had originally been described agar with concentration of substrates described previously (Cowan & Steel, 1965). Acid production from carbohydrates as Bacillus globisporus subsp. marinus by Ru$ ger & was determined as described by Leifson (1963). Growth Richter (1979). This species has been known to under anaerobic conditions was determined after incubation produce round-spore-forming cells and contain lysine in an anaerobic chamber with marine agar that was in the cell wall (Ru$ ger, 1983; Claus & Berkeley, 1986). anaerobically prepared. Growth at various NaCl concen- Nevertheless, B. marinus had rarely been included trations was investigated on marine agar or in marine broth. within Bacillus rRNA group 2 because of the lack of Growth at various temperatures was measured on marine chemotaxonomic data, e.g. the cell wall murein type agar or in marine broth at 4–55 mC. and menaquinone type (Ash et al., 1991; Rheims et al., Isolation of DNA. Chromosomal DNA was isolated and 1999). Moreover, the 16S rDNA sequence of B. purified according to a previously described method (Yoon marinus, which would have been an important criterion et al., 1996), with the exception that ribonuclease T1 was for grouping this species within rRNA group 2, was used together with ribonuclease A. not available until a recent publication by Ru$ ger et al. Chemotaxonomic characterization. Preparation of the cell (2000). In the study of Ru$ ger et al. (2000), it was also wall and determination of peptidoglycan structure were shown that the type strain of B. marinus has a cell wall carried out by the methods described by Schleifer & Kandler peptidoglycan type linked directly through -lysine. (1972) with the modification that TLC on cellulose sheets The objective of the present study was to determine the was used instead of paper chromatography. Menaquinones exact taxonomic status of B. marinus as well as that of were analysed as described previously (Komagata & Suzuki, strain YKJ-13T. On the basis of results presented, it is 1987) using reversed-phase HPLC. For quantitative analysis proposed that strain YKJ-13T should be classified as of cellular fatty acid compositions, a loop of cell mass was harvested and fatty acid methyl esters were prepared and Jeotgalibacillus alimentarius gen. nov., sp. nov., and B. identified according to the instructions of the Microbial marinus should be reclassified in Marinibacillus gen. Identification System (MIDI). nov. as Marinibacillus marinus comb. nov. DNA base composition. The DNA GjC content was determined by the method of Tamaoka & Komagata (1984). METHODS DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. Bacterial strains and culture conditions. Strain YKJ-13T was isolated from jeotgal, a traditional Korean fermented 16S rDNA sequencing and phylogenetic analysis. 16S rDNA seafood, by a dilution plating technique on marine agar was amplified by PCR using two universal primers as (Difco). B. marinus DSM 1297T, used as a reference strain, described previously (Yoon et al., 1998). The PCR product was obtained from Deutsche Sammlung von Mikro- was purified with a QIAquick PCR purification kit (Qiagen). organismen und Zellkulturen (DSMZ), Braunschweig, Sequencing of the purified 16S rDNA was performed using Germany. Cell mass for analyses of the cell wall and an ABI PRISM BigDye Terminator cycle sequencing ready menaquinones and for DNA extraction was obtained from reaction kit (Applied Biosystems) as recommended by the manufacturer. The purified sequencing reaction mixtures marine broth (Difco) culture grown at 30 mC for strain YKJ- T T were electrophoresed automatically using an Applied Bio- 13 or 20 mC for B. marinus DSM 1297 . Strains were cultivated on a horizontal shaker at 150 r.p.m. and the broth systems model 310 automatic DNA sequencer. Alignment cultures were checked for purity microscopically before of sequences was carried out with software being harvested by centrifugation. For fatty acid methyl (Thompson
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