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 by Leifson (1963). Growth under anaerobic and species descriptions below. conditions was determined after incubation in an anaerobic chamber using MA supplemented with Strain SF-20T contained meso-DAP as the diagnostic 3n1% NaCl, which was prepared anaerobically. diamino acid in the cell wall peptidoglycan. The Growth at various NaCl concentrations was investi- predominant menaquinone found in strain SF-20T was gated on MA or in MB. Growth at various tempera- unsaturated menaquinone with seven isoprene units T tures was measured on MA supplemented with 3n1% (MK-7). Strain SF-20 had a cellular fatty acid profile (w\v) NaCl at 4–55 mC. containing major amounts of branched fatty acids
2044 International Journal of Systematic and Evolutionary Microbiology 52 Lentibacillus salicampi gen. nov., sp. nov.
...... Fig. 2. Neighbour-joining tree showing the phylogenetic positions of strain SF-20T and representatives of some other related taxa based on 16S rDNA sequences. Bar, 0n01 ...... substitutions per nucleotide position. Bootstrap values T (expressed as percentages of 1000 replications) greater than Fig. 1. Light micrograph of cells of strain SF-20 from an 50% are shown at branch points. exponentially growing culture. Bar, 4 µm.
T Table 1. Cellular fatty acid profiles of strain SF-20T and The genomic DNA GjC content of strain SF-20 was related taxa 44 mol%...... T An almost complete 16S rDNA sequence of strain SF- Strains are identified as: 1, S. salexigens DSM 11483 ;2,S. 20T comprising 1521 nucleotides was determined di- marismortui DSM 12325T;3,V. pantothenticus DSM 26T;4, T T rectly after PCR amplification, representing approxi- V. proomii DSM 13055 ; 5, strain SF-20 . –, Not detected. mately 96% of the Escherichia coli 16S rRNA se- quence. In the phylogenetic tree based on the neigh- Fatty acid 12345 bour-joining algorithm, strain SF-20T was phylogeneti- cally related to the genus Bacillus and related genera Saturated fatty acids and, particularly, formed a coherent cluster with the C 0 5050505– "&:! n n n n genera Salibacillus and Virgibacillus (Fig. 2). The C 0 811100814 T "':! n n n n n clustering fidelity between strain SF-20 and the cluster C 2OH – 0 9––– "':! n comprising the genera Salibacillus and Virgibacillus C –––19– "(:! n was supported by bootstrap analysis at a confidence Unsaturated fatty acids level of 67n2%. This tree topology was also found C ω7c alcohol 1 510– – 05 "':" n n n in trees generated with the maximum-likelihood Branched fatty acids and maximum-parsimony algorithms. Strain SF-20T iso-C –06––– "":! n showed levels of 16S rDNA similarity of 93n0–94n7% anteiso-C –05––– "":! n to the genus Salibacillus and 94n0–94n1% to the genus iso-C –––12– "$:! n Virgibacillus, and levels of similarity of 92n0–93n0 and anteiso-C ––0305– "$:! n n 92n4–93n1%, respectively, to the genera Halobacillus iso-C 3 0351452120 "%:! n n n n n and Gracilibacillus. iso-C"&:! 32n934n44n932n23n4 ante-C"&:! 41n731n451n733n138n6 Many bacterial strains have been isolated from a salt iso-C"':! 4n77n66n07n130n1 field located in the Yellow Sea of Korea in our recent iso-C"(:! 3n57n62n46n50n7 study. Most of the isolates were observed to have ante-C"(:! 10n210n931n910n913n4 halotolerant or moderately halophilic physiological iso-C"(:"ω10c 0n4–––– properties, as expected. Gram-positive or -variable, Summed feature 4* 0n7–––– endospore-forming rods were found to make up a large proportion of the halotolerant or moderately * Summed features represent groups of two or three fatty acids halophilic bacterial strains isolated from the salt field. that can not be separated by GLC with the MIDI system. Among these rods, one moderately halophilic strain, Summed feature 4 contains one or more of the following fatty SF-20T, was focused on in this study, because it was acids: iso-C"(:" I and\or anteiso-C"(:" B. found to be more phylogenetically related to the recently described genera Virgibacillus, Salibacillus, Gracilibacillus and Halobacillus than to Bacillus rRNA (Table 1), with anteiso-C"&:! and iso-C"':! as the major group 1, including Bacillus subtilis (Fig. 2). In the fatty acids. The cellular polar lipids found in strain SF- phylogenetic tree based on the neighbour-joining 20T were phosphatidylglycerol (PG), diphosphatidyl- algorithm, strain SF-20T clusters with the clade in- glycerol (DPG) and some unidentified polar lipids. cluding the genera Salibacillus and Virgibacillus, sup- http://ijs.sgmjournals.org 2045 J.-H. Yoon, K. H. Kang and Y.-H. Park
Table 2. Phenotypic characteristics of strain SF-20T and related taxa ...... Taxa are identified as: 1, S. salexigens (data from Arahal et al., 2000; Garabito et al., 1997); 2, S. marismortui (Arahal et al., 1999, 2000); 3, V. pantothenticus (Heyndrickx et al., 1999; Wainø et al., 1999); 4, V. proomii (Heyndrickx et al., 1999; Wainø et al., 1999); 5, strain SF-20T. j, Positive; k, negative; , variable; , not determined. All taxa are rods and are negative for acid production from -xylose and positive for hydrolysis of casein.
Character 1 2 3 4 5
Spore shape Ellipsoidal Ellipsoidal Spherical or ellipsoidal Spherical or ellipsoidal Spherical or oval Spore position Central or Terminal or Terminal or Terminal or Terminal subterminal subterminal subterminal subterminal Anaerobic growth kk j j k NaCl range (%) 8–16 5–25 0–10 2–23 Maximum growth 45 50 50 50 40 temperature Nitrate reduction kj k Acid production from -Arabinose kj k -Cellobiose k -Fructose jj j j k -Galactose kk j j k D-Glucose jj j j k Glycerol jj j k Lactose kk kk Maltose jj j j k D-Mannitol jk k k k -Mannose jk j j k myo-Inositol kjk -Rhamnose k j k -Ribose jjk -Sorbitol kk Stachyose j Sucrose kj k -Trehalose kk j j k Hydrolysis of Aesculin j jjk Starch kk k Tween 80 kk j
Major fatty acids* iso-C"&:! iso-C"&:! iso-C"&:! or anteiso-C"(:! iso-C"&:! iso-C"':! Polar lipids† PG, DPG PG, DPG, PE PG, DPG GjC content (mol%) 40 41 37 37 44
* All taxa contain anteiso-C"&:! as a major fatty acid. † PG, Phosphatidylglycerol; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine. ported by a bootstrap resampling value of 67n2% (Fig. members of the genus Halobacillus contain -orni- 2). The relationship between this cluster and that thine, instead of meso-DAP, at position 3 of the cell comprising the genera Halobacillus and Gracilibacillus wall peptidoglycan (Spring et al., 1996) and members is supported by bootstrap analysis at a confidence level of some genera belonging to the Bacillus rRNA group of 98n8% (Fig. 2). Therefore, from the result of 2 contain -lysine or -ornithine at position 3 of the phylogenetic inference, strain SF-20T was mainly cell-wall peptidoglycan (Yoon et al., 2001). There are compared with the genera Salibacillus and Virgi- conspicuous differences between strain SF-20T and the bacillus to determine its exact taxonomic position. genera Salibacillus and Virgibacillus in cellular fatty Chemotaxonomic properties do not necessarily allow acid profiles, particularly in the proportion of the fatty T clear differentiation between aerobic or facultatively acid iso-C"':!. Strain SF-20 and all members of the anaerobic, rod-shaped, endospore-forming genera. genera Salibacillus and Virgibacillus contain major Most of these genera contain MK-7 as the predomi- amounts of anteiso-C"& ! (Table 1). However, strain T : nant menaquinone and meso-DAP as the diamino acid SF-20 has a significant amount of iso-C"':!, which is at position 3 of the cell-wall peptidoglycan. However, a minor component in the genera Salibacillus and
2046 International Journal of Systematic and Evolutionary Microbiology 52 Lentibacillus salicampi gen. nov., sp. nov.
Virgibacillus. Strain SF-20T also shows noteworthy adonitol, -arabinose, -cellobiose, -fructose, -ga- differences from members of the genera Salibacillus lactose, -glucose, lactose, maltose, -mannitol, - and Virgibacillus in the amounts of two other fatty mannose, -melezitose, melibiose, myo-inositol, - T acids, iso-C"%:! and iso-C"&:! (Table 1). Strain SF-20 raffinose, -rhamnose, -ribose, -sorbitol, sucrose, - shows a difference from the genus Virgibacillus in its trehalose or -xylose. The GjC content of the type polar lipid pattern. The genus Virgibacillus shows the strain is 44 mol% (determined by HPLC). The type T T presence of phosphatidylethanolamine, which is not strain, strain SF-20 (l KCCM 41560 l JCM found in the genus Salibacillus or in strain SF-20T 11462T), was isolated from a salt field of the Yellow (Wainø et al., 1999). Growth of strain SF-20T was Sea in Korea. found to be obviously slow when compared with growth of members of the genera Salibacillus and Acknowledgements Virgibacillus. Accordingly, it appears to be appropriate that strain SF-20T be classified in a new genus separate This work was supported by grant HSS0310134 and the from all meso-DAP-containing bacilli as well as the NRL research program (grants M10104000294- genera Salibacillus and Virgibacillus. Therefore, on the 01J000012800 and NLW0070111) from the Ministry of basis of the phenotypic and phylogenetic data, we Science and Technology (MOST) of the Republic of Korea and by the research fund of the Probionic Corporation of propose the creation of a novel genus and species, Korea. Lentibacillus salicampi gen. nov., sp. nov., for strain SF-20T. Characters that are useful in the differentiation of the novel taxon from members of related genera are References ! shown in Table 2. Arahal, D. R., Marquez, M. C., Volcani, B. E., Schleifer, K. H. & Ventosa, A. (1999). Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int J Syst Bacteriol 49, 521–530. Description of Lentibacillus gen. nov. ! Arahal, D. R., Marquez, M. C., Volcani, B. E., Schleifer, K. H. & Lentibacillus (Len.ti.ba.cilhlus. L. adj. lentus slow; L. Ventosa, A. (2000). Reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov. Int J Syst Evol Microbiol 50, dim. n. bacillus small rod; N.L. masc. n. Lentibacillus 1501–1503. slowly growing bacillus). Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872, AL Cells are rods. Gram-variable. Spherical or oval 174 .InBergey’s Manual of Systematic Bacteriology, vol. 2, pp. endospores lie terminally in swollen sporangia. Motile. 1105–1139. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins. Colonies are cream-coloured, smooth, circular to Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of slightly irregular and raised on MA supplemented with Medical Bacteria. London: Cambridge University Press. 3 1% NaCl. Catalase- and oxidase-positive. Urease- n Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a negative. The cell wall peptidoglycan contains meso- maximum likelihood approach. J Mol Evol 17, 368–376. DAP at position 3 of the peptide subunit. The Felsenstein, J. (1993). : phylogenetic inference package, version predominant menaquinone is MK-7. The major fatty 3.5. Distributed by the author. Department of Genetics, University of acids are anteiso-C and iso-C . The G C con- Washington, Seattle, USA. "&:! "':! j ! tent of the type strain of the type species is 44 mol% Garabito, M. J., Arahal, D. R., Mellado, E., Marquez, M. C. & (determined by HPLC). The type species is Lenti- Ventosa, A. (1997). Bacillus salexigens sp. nov., a new moderately bacillus salicampi. halophilic Bacillus species. Int J Syst Bacteriol 47, 735–741. 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