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 et al., 1994). Gaps at the 5h and 3h ends of the ester analysis, cell mass of strain YKJ-13T and B. marinus alignment were omitted from further analysis. Phylogenetic T trees were inferred using three tree-making algorithms, i.e. DSM 1297 was obtained after growth for 3 d at 30 mCon marine agar. the neighbour-joining (Saitou & Nei, 1987), maximum- likelihood (Felsenstein, 1981) and maximum-parsimony Morphological characterization. The morphologies of col- (Kluge & Farris, 1969) methods in the  package onies and cells grown on marine agar were examined. Cell (Felsenstein, 1993). Evolutionary distance matrices for the micromorphology was observed using phase-contrast mi- neighbour-joining method were calculated with the algor- croscopy and transmission electron microscopy. Flagellum ithm of Jukes & Cantor (1969) with the  program. type was determined using transmission electron microscopy The stability of relationships was assessed by a bootstrap with cells from an exponentially growing culture. For analysis based on 1000 resamplings of the neighbour-joining transmission electron microscopic observation, cells were dataset using the programs  and  of the negatively stained with 1% (w\v) phosphotungstic acid and,  package.

2088 International Journal of Systematic and Evolutionary Microbiology 51 Jeotgalibacillus alimentarius gen. nov., sp. nov.

RESULTS incubation on marine agar, colonies were smooth, Morphology glistening, irregular, flat-raised and orange-yellow in colour. Most cells of strain YKJ-13T were rods measuring 1n0–1n2i2n0–4n0 µm in 3 d culture on marine agar at Cultural and physiological characteristics 30 mC; however, longer rods were often observed. Cells T were Gram-variable and motile by means of peri- Strain YKJ-13 grew optimally at 30–35 mC. Growth trichous flagella. Round spores were located sub- occurred at 10 and 45 mC, but not at 4 or 50 mC. terminally or terminally in swollen sporangia. After It grew optimally at pH 7n0–8n0 and no growth was

Table 1. Differential phenotypic characteristics of strain YKJ-13T and B. marinus ...... j, Positive reaction; k, negative reaction; j, weakly positive reaction; , variable reaction. Both species were rod-shaped, produced round spores (terminal or subterminal), had peritrichous flagella and were positive for motility, catalase, decomposition of aesculin and gelatin, and acid production from glucose, fructose, maltose, sucrose and trehalose. Both species were negative for decomposition of starch and urea, and acid production from adonitol, arabinose, cellobiose, rhamnose and sorbitol.

Characteristic Strain YKJ-13T B. marinus*

Swollen sporangia jkor slightly Gram staining  j Pigment jk Anaerobic growth jk Cytochrome oxidase j  (k) Nitrate reduction j  (k) Decomposition of: Casein j  (j) Hypoxanthine kk† Tween 80 jk† Tyrosine kk† Xanthine kk† Acid production from: Lactose k  (j) Xylose k  (j) Galactose jk Mannitol jk Mannose kj Melibiose jk Raffinose jk Growth in NaCl at: 0% kk 7% j  (j) 10% jk 20% jk 21% kk Growth at (mC): 5 kj 10 jj 30 j  (j) 37 jk Optimum growth temperature (mC) 30–35 12–23 Maximum growth temperature (mC) 45–50 25–30 Peptidoglycan type -Lys-direct -Lys-direct† Predominant menaquinone MK-7, MK-8 MK-7† Major fatty acid iso-C"&:! anteiso-C"&:!† GjC content (mol%) 44 37–42

* Data from Ru$ ger & Richter (1979), Ru$ ger (1983) and Ru$ ger et al. (2000), except where indicated. Data in parentheses are for the type strain of B. marinus from Ru$ ger & Richter (1979), Ru$ ger (1983) or Ru$ ger et al. (2000). † Data are for B. marinus DSM 1297T obtained from this study.

International Journal of Systematic and Evolutionary Microbiology 51 2089 J.-H. Yoon and others

Table 2. Cellular fatty acid profiles of strain YKJ-13T and B. marinus DSM 1297T on marine agar

Strain Fatty acid composition (%)*

i-C14:0 C14:0 i-C15:0 ai-C15:0 C15:0 C16:1 ω7c alcohol C16:1 ω11c i-C16:0 C16:0 i-C17:1 ω10c i-C17:0 ai-C17:0 C18:0 Summed feature 5†

T Strain YKJ-13 2n10n747n615n21n15n21n21n93n93n86n54n63n42n8 B. marinus 5n3  22n247n7  2n6  5n11n5  2n610n7  2n3 DSM 1297T

, Not detected. * i, iso; ai, anteiso. † Summed feature 5 represents iso-C"(:" I and\or anteiso-C"(:" B, which could not be separated by GLC with the MIDI system.

...... Fig. 1. Neighbour-joining tree showing the phylogenetic position of strain YKJ-13T, some Bacillus spp. and representatives of some other taxa based on 16S rDNA sequences. Bar, 0n01 substitution per nucleotide position. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at the branch points.

2090 International Journal of Systematic and Evolutionary Microbiology 51 Jeotgalibacillus alimentarius gen. nov., sp. nov.

T observed at pH values below 6n0. Strain YKJ-13 resampling value of 91n8%. The phylogenetic rela- grew optimally in the presence of 3–12% (w\v) NaCl. tionship between strain YKJ-13T, B. marinus and It grew in the presence of 19% (w\v) NaCl and weakly members of Bacillus rRNA group 2 was also found in the presence of 20% NaCl. Growth did not occur in trees generated by the maximum-likelihood and in the presence of more than 21% (w\v) NaCl. Strain maximum-parsimony algorithms. Strain YKJ-13T YKJ-13T grew under anaerobic conditions on marine showed the highest degree of relatedness to B. marinus T T agar. Strain YKJ-13 showed catalase and oxidase DSM 1297 , sharing 95n7% 16S rDNA similarity. activities, but no urease activity. Aesculin, casein, Sequence similarity values of 16S rDNA from strain gelatin and Tween 80 were hydrolysed; no hydrolysis YKJ-13T with 16S rDNA from the type strains of of hypoxanthine, starch, tyrosine or xanthine was Bacillus species belonging to rRNA group 2 were in T observed. Nitrate was reduced to nitrite. Phenotypic the range 92n4–93n9%. Strain YKJ-13 exhibited levels T properties of strain YKJ-13 are summarized in of 16S rDNA similarity of less than 94n2% with the Table 1, together with those of B. marinus. type strains of other Bacillus species that had known 16S rDNA sequences. Chemotaxonomic characteristics and DNA base composition DISCUSSION No diaminopimelic acid was found as the diagnostic Many bacilli have been recently isolated from jeotgal. amino acid in the cell wall hydrolysate of strain YKJ- T T In the course of characterizing these strains, one isolate 13 . The cell wall of strain YKJ-13 contained a (strain YKJ-13T) has attracted our attention because directly cross-linked peptidoglycan with -lysine as the of its morphological characteristic of forming round diagnostic diamino acid. On the basis of this result, it endospores and its moderately halophilic physiology. was concluded that the peptidoglycan type of strain T 16S rDNA sequence analysis showed that strain YKJ- YKJ-13 is A1α, based directly on -Lys, as described 13T was phylogenetically related to the radiation of a by Schleifer & Kandler (1972). Dinitrophenylated cell T cluster comprising members of Bacillus rRNA group walls of strain YKJ-13 contained only ε-DNP-lysine, 2, with the formation of a coherent cluster with B. thus confirming the above-mentioned type. B. marinus T T marinus (Fig. 1). Strain YKJ-13 contained -lysine at DSM 1297 had a directly cross-linked peptidoglycan position 3 of the cell wall peptidoglycan, which is based on -lysine as the diamino acid. The α-carboxyl characteristic of Bacillus rRNA group 2 (Rheims et al., group of -glutamic acid was substituted by a glycine 1999; Stackebrandt et al., 1987). Strain YKJ-13T had residue. This result also has recently been proven by MK-7 and MK-8 as the predominant menaquinones Ru$ ger et al. (2000). To our knowledge, this type has and iso-C"&:! as the major fatty acid. Among the not been found in any other organism up to now. T rRNA group 2 bacilli, some species, such as Bacillus Strain YKJ-13 contained MK-7 (62%) as the pre- sphaericus and Bacillus silvestris, have been found to dominant menaquinone and also a significant amount T contain iso-C"&:! as the major fatty acid (Rheims et al., of MK-8 (35%). B. marinus DSM 1297 contained a 1999; Shida et al., 1997). On the basis of these data, large proportion of MK-7 (87%). The cellular fatty T T strain YKJ-13 is considered to be a member of acid profile of strain YKJ-13 was characterized by the Bacillus rRNA group 2. Results showed that the type predominance of iso-C"& ! (47n6%) followed by : T strain of B. marinus had MK-7 as the predominant anteiso-C"&:! (15n2%), whereas B. marinus DSM 1297 menaquinone and -lysine at position 3 of the peptide was characterized by the predominance of anteiso- subunit of the peptidoglycan. B. marinus has already C"&:! (47n7%) and the occurrence of significant been known to form round endospores (Ru$ ger, 1983; amounts of iso-C"&:! (22n2%) and anteiso-C"(:! Claus & Berkeley, 1986). Accordingly, B. marinus (10n7%) (Table 2). The DNA GjC content of strain must also be a member of Bacillus rRNA group 2. YKJ-13T was 44 mol%. 16S rDNA sequence comparison showed that strain T Phylogenetic analysis YKJ-13 and the type strain of B. marinus exhibited the closest phylogenetic relationship, with 95n7% The 16S rDNA of strain YKJ-13T was directly sequen- similarity. There were some physiological differences ced after PCR amplification and its almost complete between strain YKJ-13T and B. marinus, such as the nucleotide sequence was determined. This sequence ability to grow anaerobically, and NaCl and growth comprised 1508 nt, representing approximately 96% temperature tolerances (Table 1). Whereas B. marinus of the Escherichia coli 16S rRNA sequence. In the tree- showed slight growth at 7% NaCl and no growth at based neighbour-joining algorithm, strain YKJ-13T 10% NaCl, strain YKJ-13T grew at 20% NaCl (Table was phylogenetically related to members of Bacillus 1). Strain YKJ-13T is a mesophile growing at 10 and rRNA group 2 and formed a coherent cluster with 45 mC with an optimum of 30–35 mC, whereas B. B. marinus DSM 1297T (Fig. 1). The clustering fidelity marinus is a psychrophile showing a growth range of T T between strain YKJ-13 and B. marinus DSM 1297 5–30 mC, with an optimum of 12–23 mC (Table 1). was supported by bootstrap analysis at a confidence Some chemotaxonomic properties are important for T level of 93n6%. The relationship between this cluster distinguishing strain YKJ-13 from B. marinus.Itis and the cluster comprising members of Bacillus rRNA noteworthy that the fatty acid profile of strain YKJ- group 2 was supported by a relatively high bootstrap 13T differed from that of B. marinus DSM 1297T.

International Journal of Systematic and Evolutionary Microbiology 51 2091 J.-H. Yoon and others

T T Strain YKJ-13 and B. marinus DSM 1297 showed a are MK-7 and MK-8. Major fatty acid is iso-C"&:!. distinct difference in the amounts of iso-C"&:! and DNA GjC content is 44 mol% (determined by anteiso-C"&:! they contained (Table 2). Strain YKJ- HPLC). Type species is Jeotgalibacillus alimentarius. 13T could be distinguished from the type strain of B. marinus by menaquinone composition. The mena- Description of Jeotgalibacillus alimentarius sp. nov. quinone profile of strain YKJ-13T was characterized by the predominance of MK-7 (62%) followed by Jeotgalibacillus alimentarius (a.li.men.tahri.us. L. adj. MK-8 (35%), whereas B. marinus DSM 1297T has alimentarius relating to food). 87% MK-7 and no MK-8. This menaquinone profile T Gram-variable, facultatively anaerobic rods of 1n0– of strain YKJ-13 has not been found in other bacilli 1n2i2n0–4n0 µm; longer rods are often observed. belonging to rRNA group 2 (Rheims et al., 1999). B. T Round endospores lie subterminally or terminally in marinus DSM 1297 has a unique peptidoglycan swollen sporangia. Motile by means of peritrichous structure that has not been found in any other flagella. Colonies are smooth, glistening, irregular, flat organism. On the basis of these data, particularly T to raised and orange-yellow in colour on marine agar. chemotaxonomic data, strain YKJ-13 and B. marinus Grows in the presence of 19% NaCl and weakly in the should be distinguished as members of different genera presence of 20% NaCl. Growth occurs at 10 and in Bacillus rRNA group 2. 45 mC, but not at 4 or 50 mC. Optimal growth tem- The taxonomic status of the rRNA group 2 bacilli has perature is 30–35 mC. Optimal pH for growth is pH recently been discussed. It has been recognized that 7n0–8n0 and no growth is observed at pH 6n0. Catalase- Bacillus species belonging to this group should be and oxidase-positive. Urease-negative. Aesculin, taxonomically distinguished from Bacillus rRNA casein, gelatin and Tween 80 are hydrolysed; no group 1, including Bacillus subtilis, the type species of hydrolysis of hypoxanthine, starch, tyrosine or xan- this genus, and all meso-diaminopimelic acid-contain- thine is observed. Nitrate is reduced to nitrite. Acid is ing bacilli by micromorphology, phylogeny and cell produced from -ribose, -fructose, -mannitol, meli- wall composition (Farrow et al., 1994). Therefore, the biose, -melezitose, -glucose, -galactose, sucrose, -lysine- and -ornithine-containing bacilli of rRNA maltose, -trehalose and -raffinose. Acid is not group 2 may be in need of taxonomic re-evaluation to produced from -sorbitol, myo-inositol, -xylose, - transfer them into several new genera or existing arabinose, -mannose, adonitol, -rhamnose, - genera. Since no proposal about taxonomic reclassi- cellobiose, stachyose or lactose. Peptidoglycan type is fication of the rRNA group 2 bacilli has been A1α (-Lys-direct). Predominant menaquinones are forwarded, Rheims et al. (1999) proposed that any MK-7 and MK-8. Major fatty acid is iso-C"&:!. DNA strain worthy of species rank within rRNA group 2, GjC content is 44 mol% (determined by HPLC). Isolated from jeotgal, traditional Korean fermented exhibiting taxonomic properties that would identify T Bacillus seafood. Type strain is strain YKJ-13 ( l KCCM this taxon as a member of the genus , should T T still be described as a species of this genus. However, 80002 l JCM 10872 ). there is no longer any reason to delay taxonomic re-evaluation of the rRNA group 2 bacilli. Recently, Description of Marinibacillus gen. nov. B. globisporus, Bacillus psychrophilus and Bacillus pasteurii, which belong to rRNA group 2, together Marinibacillus (Ma.ri.ni.ba.cilhlus. L. adj. marinus of with one novel isolate, were transferred to the genus the sea; Gr. n. baktron rod; M.L. masc. n. Marini- Sporosarcina (Yoon et al., 2001). Accordingly, it bacillus rod of the sea). T appears to be more appropriate that strain YKJ-13 The description is the same as that given by Ru$ ger and B. marinus be placed within two other genera, not (1983) and Ru$ ger et al. (2000). Additional chemo- the genus Bacillus. taxonomic characters found in this study are as Therefore, the creation of a new genus and species, follows: cell wall peptidoglycan contains -lysine at Jeotgalibacillus alimentarius gen. nov., sp. nov., for position 3 of the peptide subunit; the predominant strain YKJ-13T and the reclassification of B. marinus menaquinone is MK-7; and the major fatty acid is (Ru$ ger 1983) in a new genus, Marinibacillus gen. nov., anteiso-C"&:!. Type species is Marinibacillus marinus. as Marinibacillus marinus comb. nov. are proposed. Description of Marinibacillus$ marinus comb. nov. Description of Jeotgalibacillus gen. nov. (basonym Bacillus marinus Ruger 1983) Jeotgalibacillus jeotgal The species description is as given above for the genus. (Je.ot.ga.li.ba.cilhlus. Korean n. T jeotgal, traditional Korean food; Gr. n. baktron rod; Type strain is DSM 1297 . N.L. masc. n. Jeotgalibacillus rod from jeotgal). Rod-shaped bacterium. Round endospores are ob- ACKNOWLEDGEMENTS served in swollen sporangia. Catalase- and oxidase- This work was supported by grants HS2701 and positive. Urease-negative. Nitrate is reduced to nitrite. HSS0310033 from the Ministry of Science and Technology Cell wall peptidoglycan contains -lysine at position 3 (MOST) of the Republic of Korea and by the research fund of the peptide subunit. Predominant menaquinones of the Probionic Corporation of Korea.

2092 International Journal of Systematic and Evolutionary Microbiology 51 Jeotgalibacillus alimentarius gen. nov., sp. nov.

$ REFERENCES Ruger, H.-J. & Richter, G. (1979). Bacillus globisporus subsp. marinus subsp. nov. Int J Syst Bacteriol 29, 196–203. Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). $ $ Phylogenetic heterogeneity of the genus Bacillus as revealed by Ruger, H.-J, Fritze, D. & Sproer, C. (2000). New psychrophilic and comparative analysis of small-subunit-ribosomal RNA psychrotolerant Bacillus marinus strains from tropical and sequences. Lett Appl Microbiol 13, 202–206. polar deep-sea sediments and emended description of the species. Int J Syst Evol Microbiol 50, 1305–1313. Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872. In Bergey’s Manual of Systematic Bacteriology, vol. 2, pp. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a 1105–1140. Edited by P. H. A. Sneath, N. S. Mair, M. E. new method for reconstructing phylogenetic trees. Mol Biol Sharpe & J. G. Holt. Baltimore: Williams & Wilkins. Evol 4, 406–425. Clausen, V., Jones, J. G. & Stackebrandt, E. (1985). 16S ribosomal Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of RNA analysis of Filibacter limicola indicates a close relationship bacterial cell walls and their taxonomic implications. Bacteriol to the genus Bacillus. J Gen Microbiol 131, 2659–2663. Rev 36, 407–477. Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, Medical Bacteria. London: Cambridge University Press. K. (1997). Transfer of Bacillus alginolyticus, Bacillus chon- droitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Farrow, J. A. E., Ash, C., Wallbanks, S. & Collins, M. D. (1992). Bacillus kobensis, and Bacillus thiaminolyticus to the genus Phylogenetic analysis of the genera Planococcus, Marinococcus Paenibacillus and emended description of the genus Paeni- and Sporosarcina and their relationships to members of the bacillus. Int J Syst Bacteriol 47, 289–298. genus Bacillus. FEMS Microbiol Lett 93, 167–172. Stackebrandt, E., Ludwig, W., Weizenegger, M., Dorn, S., McGill, Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1994). Phylo- T. J., Fox, G. E., Woese, C. R., Schubert, W. & Schleifer, K.-H. genetic interrelationships of round-spore-forming bacilli (1987). Comparative 16S rRNA oligonucleotide analyses and containing cell walls based on lysine and the non-spore- murein types of round-spore-forming bacilli and non-spore- forming genera Caryophanon, Exiguobacterium, Kurthia, and forming relatives. J Gen Microbiol 133, 2523–2529. Planococcus. Int J Syst Bacteriol 44, 74–82. Tamaoka, J. & Komagata, K. (1984). Determination of DNA Felsenstein, J. (1981). Evolutionary trees from DNA sequences: base composition by reverse-phase high-performance liquid a maximum likelihood approach. J Mol Evol 17, 368–376. chromatography. FEMS Microbiol Lett 25, 125–128. Felsenstein, J. (1993).  (phylogenetic inference package), Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).  version 3.5. Department of Genetics: University of Washing- : improving the sensitivity of progressive multiple sequence ton, Seattle, USA. alignment through sequence weighting, position-specific gap Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein penalties and weight matrix choice. Nucleic Acids Res 22, molecules. In Mammalian Protein Metabolism, vol. 3, pp. 4673–4680. 21–132. Edited by H. N. Munro. New York: Academic Press. Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharo- evolution of anurans. Syst Zool 18, 1–32. monospora strains by the use of genomic DNA fragments and Komagata, K. & Suzuki, K. (1987). Lipids and cell-wall analysis in rRNA gene probes. Int J Syst Bacteriol 46, 502–505. bacterial systematics. Methods Microbiol 19, 161–203. Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific Lanyi, B. (1987). Classical and rapid identification methods for phylogenetic analysis of the genus Nocardioides and related medically important bacteria. Methods Microbiol 19, 1–67. taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194. Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184. Yoon, J.-H., Lee, K.-C., Weiss, N., Kho, Y. H., Kang, K. H. & Park, $ Y.-H. (2001). Sporosarcina aquimarina sp. nov., a bacterium Rheims, H., Fruhling, A., Schumann, P., Rohde, M. & isolated from a seawater in Korea, and transfer of Bacillus Stackebrandt, E. (1999). Bacillus silvestris sp. nov., a new globisporus (Larkin & Stokes 1967), Bacillus psychrophilus member of the genus Bacillus that contains lysine in its cell wall. (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the Int J Syst Bacteriol 49, 795–802. $ genus Sporosarcina as Sporosarcina globispora comb. nov., Ruger, H.-J. (1983). Differentiation of Bacillus globisporus, Sporosarcina psychrophila comb. nov. and Sporosarcina Bacillus marinus comb. nov., Bacillus aminovorans, and Bacillus pasteurii comb. nov., and emended description of the genus insolitus. Int J Syst Bacteriol 33, 157–161. Sporosarcina. Int J Syst Evol Microbiol 51, 1079–1086.

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