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Lentibacillus Salicampi Gen. Nov., Sp. Nov., a Moderately Halophilic

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Lentibacillus Salicampi Gen. Nov., Sp. Nov., a Moderately Halophilic International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2043–2048 DOI: 10.1099/ijs.0.02335-0 Lentibacillus salicampi gen. nov., sp. nov., a NOTE moderately halophilic bacterium isolated from a salt field in Korea 1 Microbial Genomics Jung-Hoon Yoon,1 Kook Hee Kang2 and Yong-Ha Park1,3 Laboratory, Korea Research Institute of Bioscience and Author for correspondence: Biotechnology (KRIBB), Yong-Ha Park. Tel: j82 42 860 4620. Fax: j82 42 862 1315. PO Box 115, Yusong, e-mail: yhpark!mail.kribb.re.kr Taejon, Korea 2 Department of Food and A Gram-variable, aerobic, endospore-forming, rod-shaped bacterial strain, SF- Life Science, 20T, which was isolated from a salt field in Korea, was subjected to a Sungkyunkwan University, Chunchun-dong 300, polyphasic taxonomic study. Cells of this organism were motile by means of Jangan-gu, Suwon, Korea single flagella. Strain SF-20T grew optimally in the presence of 4–8% NaCl. The 3 National Research cell wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic Laboratory of Molecular diamino acid. The predominant menaquinone is MK-7. Strain SF-20T has a Ecosystematics, Institute of cellular fatty acid profile containing major amounts of branched fatty acids. Probionics, Probionic Corporation, Bio-venture The major fatty acids are anteiso-C15:0 and iso-C16:0. The cellular phospholipids Centre, KRIBB, Yusong, are phosphatidylglycerol and diphosphatidylglycerol. The GMC content of the Taejon, Korea DNA is 44 mol%. Strain SF-20T is phylogenetically closely related to the genus Bacillus and some related genera and, particularly, formed a coherent cluster with the genera Salibacillus and Virgibacillus. The clustering fidelity between strain SF-20T and the cluster comprising these two genera was supported by bootstrap analysis at a confidence level of 672%. Strain SF-20T exhibited levels of 16S rDNA similarity of 930–947% to the genus Salibacillus and 940–941% to the genus Virgibacillus. On the basis of phenotypic and phylogenetic data, strain SF-20T should be classified in a novel genus and species, for which the name Lentibacillus salicampi gen. nov., sp. nov. is proposed. The type strain is strain SF-20T (l KCCM 41560T l JCM 11462T). Keywords: genus Lentibacillus, Lentibacillus salicampi sp. nov., salt field, moderate halophile Aerobic or facultatively anaerobic, endospore-forming comparative phenotypic analyses have revealed that it rods are widely distributed in nature (Claus & may be more appropriate to place some of these bacilli Berkeley, 1986; Slepecky & Hemphill, 1991). Mod- in new genera or other related genera. Therefore, some erately halophilic and halotolerant endospore-forming of these species have been reclassified as members of rods have commonly been isolated from marine new genera such as Virgibacillus (Heyndrickx et al., environments and related regions or materials 1998), Salibacillus (Wainø et al., 1999) and Marini- (Ventosa et al., 1998). For a long time, most of these bacillus (Yoon et al., 2001). In addition, some new bacteria were assigned to the genus Bacillus, e.g. genera, for example Gracilibacillus (Wainø et al., 1999) Bacillus halophilus, Bacillus marinus, Bacillus maris- and Halobacillus (Spring et al., 1996), have been mortui, Bacillus salexigens, Bacillus dipsosauri, Bacillus proposed to affiliate some newly isolated moderately pantothenticus and so on (Arahal et al., 1999; Claus & halophilic and halotolerant endospore-forming rods. Berkeley, 1986; Garabito et al., 1997; Ventosa et al., B. dipsosauri and B. marismortui have recently re- 1989). However, 16S rRNA sequence analyses and spectively been transferred to the genera Gracilibacillus Salibacillus et al et al ................................................................................................................................................. and (Arahal ., 2000; Wainø ., Abbreviations: DAP, diaminopimelic acid; FAME, fatty acid methyl ester; 1999). MA, marine agar; MB, marine broth; TEM, transmission electron mi- croscopy. In this study, we describe a Gram-variable, endospore- T forming, moderately halophilic bacterial strain, SF- The GenBank accession number for the 16S rDNA sequence of strain SF-20 T is AY057394. 20 , isolated from a salt field of the Yellow Sea in 02335 # 2002 IUMS Printed in Great Britain 2043 J.-H. Yoon, K. H. Kang and Y.-H. Park Korea. From the results of 16S rDNA sequence Chromosomal DNA was isolated and purified ac- comparison, this organism was found to be phylo- cording to the method described previously (Yoon et genetically related to the genera Virgibacillus, Sali- al., 1996), with the exception that ribonuclease T1 was bacillus, Gracilibacillus and Halobacillus. Accordingly, used together with ribonuclease A. The isomer type of the aim of this work was to establish the exact diaminopimelic acid (DAP) in the peptidoglycan was taxonomic position of strain SF-20T with a com- determined by the method of Komagata & Suzuki bination of phenotypic characters and detailed phylo- (1987). Menaquinones were analysed as described genetic analysis. On the basis of the data presented previously (Komagata & Suzuki, 1987) using reverse- below, we propose that strain SF-20T should be phase HPLC. Polar lipids were extracted using the classified as a novel genus and species, Lentibacillus procedures described by Minnikin et al. (1984) and salicampi gen. nov., sp. nov. identified by two-dimensional TLC followed by spray- ing with appropriate detection reagents (Komagata & Strain SF-20T was isolated by the dilution plating Suzuki, 1987). For quantitative analysis of cellular technique on marine agar (MA) (Difco) supplemented fatty acid compositions, a loop of cell mass was with 8 1% (w\v) NaCl. Cell biomass for the analyses n harvested and FAMEs were prepared and identified of cell wall, menaquinones and polar lipids and for according to the instructions of the Microbial Identi- DNA extraction was produced in marine broth (MB) fication System (MIDI). The G C content was (Difco) supplemented with 3 1%(w\v) NaCl at 30 C. j n m determined by the method of Tamaoka & Komagata Strain SF-20T was cultivated on a horizontal shaker at (1984). DNA was hydrolysed and the resultant nucleo- 150 r.p.m. and the broth cultures were checked micro- tides were analysed by reverse-phase HPLC. scopically for purity before being harvested by centri- fugation. For fatty acid methyl ester (FAME) analysis, T 16S rDNA was amplified by PCR using two universal cell mass of strain SF-20 and some reference strains primers as described previously (Yoon et al., 1998). was obtained from agar plates after growing for 7 days The PCR product was purified with a QIAquick PCR at 30 mC on MA. The reference strains included T purification kit (Qiagen). Sequencing of the purified Salibacillus salexigens DSM 11483 , Salibacillus maris- 16S rDNA was performed using an ABI PRISM mortui DSM 12325T, Virgibacillus pantothenticus DSM T T BigDye Terminator cycle sequencing ready reaction 26 and Virgibacillus proomii DSM 13055 and were kit (Applied Biosystems) as recommended by the obtained from the DSMZ. manufacturer. The purified sequencing reaction mix- Colony and cell morphologies were examined by using tures were electrophoresed automatically using an colonies grown on MA supplemented with 3n1%(w\v) Applied Biosystems model 310 automatic DNA se- NaCl. Observation of cell morphology was performed quencer. Alignment of sequences was carried out with using light microscopy and transmission electron software (Thompson et al., 1994). Gaps at microscopy (TEM). Flagellum type was determined the 5h and 3h ends of the alignment were omitted from using TEM with cells from exponentially growing further analysis. Phylogenetic trees were inferred by culture. For TEM observation, cells were negatively using three tree-making algorithms, the neighbour- stained with 1% (w\v) phosphotungstic acid and, after joining (Saitou & Nei, 1987), maximum-likelihood air drying, the grids were examined by using a model (Felsenstein, 1981) and maximum-parsimony (Kluge CM-20 TEM (Philips). Gram reaction was determined & Farris, 1969) methods in the package using the bioMe! rieux Gram stain kit according to the (Felsenstein, 1993). Evolutionary distance matrices for manufacturer’s instructions. Oxidase activity was de- the neighbour-joining method were calculated with the termined by oxidation of 1% p-aminodimethylaniline algorithm of Jukes & Cantor (1969) with the program oxalate. Catalase activity was determined by bubble . The stability of relationships was assessed by production in a 3% (v\v) hydrogen peroxide solution. a bootstrap analysis based on 1000 resampling of the Urease activity was determined according to the neighbour-joining dataset by using the programs method of Cowan & Steel (1965) with addition of 4% and of the package. NaCl. Hydrolysis of casein and starch was determined Strain SF-20T was Gram-variable. Cells of strain SF- T as described by Cowan & Steel (1965). Hydrolysis of 20 were rods, approximately 0n4–0n7 µm wide by aesculin, hypoxanthine, Tween 80, tyrosine and xan- 2n0–4n0 µm long in a 7-day culture at 30 mConMA thine was performed on MA supplemented with 3n1% supplemented with 3n1% NaCl (Fig. 1). Growth of (w\v) NaCl with the concentrations of substrates strain SF-20T was much slower than that of members described previously (Cowan & Steel, 1965). Acid of the genera Salibacillus and Virgibacillus. Other production from carbohydrates was determined as characteristics of the novel strain are given in the genus described
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