International Journal of Systematic and Evolutionary Microbiology (2002), 52, 987–993 DOI: 10.1099/ijs.0.02033-0

Okibacterium fritillariae gen. nov., sp. nov., a novel genus of the family

1 VKM – All-Russian Lyudmila I. Evtushenko,1,2 Lubov V. Dorofeeva,1 Valentina I. Krausova,1 Collection of 2 3 4 Microorganisms, G. K. Ekaterina Yu. Gavrish, Svetlana G. Yashina and Mariko Takeuchi Skryabin Institute of Biochemistry and Physiology of Author for correspondence: Lyudmila I. Evtushenko. Tel: j7 095 925 74 48. Fax: j7 095 956 33 70. Microorganisms, Russian e-mail: evtushenko!ibpm.serpukhov.ru Academy of Sciences, Pushchino, Moscow Region, 142290, Russia Okibacterium fritillariae gen. nov., sp. nov. (type strain VKM Ac-2059T l IFO 2 Pushchino State University, 16404T) is proposed for aerobic, oxidase- and catalase-positive, coryneform Pushchino, Moscow isolated from seeds of Fritillaria ruthenica Wikstr. and Clematis recta Region, 142290, Russia L. Strains of the new genus form a distinct branch within the phylogenetic 3 Institute of Cell Biophysics, cluster composed of members of the family Microbacteriaceae and are Russian Academy of Sciences, Pushchino, characterized by B-type peptidoglycan containing amino acids glycine, Moscow Region, 142290, glutamate, homoserine, alanine and lysine, the glycolyl type of muramic acid, Russia the major menaquinones MK-10 and MK-11, the principal phospholipids 4 Institute for Fermentation, phosphatidylglycerol and diphosphatidylglycerol, and a DNA GMC content of Osaka, 17-85, Juso- approximately 67 mol%. honmachi 2-home, Yudogava-ku, Osaka 532, Japan Keywords: Actinomycetales, Okibacterium fritillariae gen. nov., sp. nov., Fritillaria ruthenica Wikstr., Clematis recta L., plant seeds

INTRODUCTION in peptidoglycan amino acid composition but differed from this genus by the major menaquinones. In this The family Microbacteriaceae proposed by Park et al. work, we present the taxonomic characterization of (1993) currently contains 12 genera which are charac- these strains and propose a new genus and a new terized by B-type peptidoglycan (Schleifer & Kandler, , Okibacterium fritillariae gen. nov., sp. nov. 1972) and unsaturated major menaquinones (Collins & Jones, 1981). Most genera of the family have peptidoglycan based upon 2,4-diaminobutyric acid or METHODS ornithine (Casida, 1986; Davis et al., 1984; Zgurskaya Isolation and cultivation of bacteria. The plants Fritillaria et al., 1993; Komagata & Suzuki, 1986; Groth et al., ruthenica Wikstr. and Clematis recta L. at the stage of 1996; Takeuchi et al., 1996; Suzuki et al., 1997; mature seeds were collected in Prioksko-Terrasny Biosphere Takeuchi & Hatano, 1998a, b; Matsuyama et al., Park, Moscow Region, Russia. For isolation of micro- 1999; Schumann et al., 1999; Ma$ nnisto$ et al., 2000; organisms, the seeds were cut into pieces, added to 1 ml Behrendt et al., 2001; Evtushenko et al., 2000, 2001). 0n85% (w\v) NaCl solution, and ground with a pestle. One Only seven of the 31 validly described species of the drop of this suspension was plated onto corynebacterial genus (Takeuchi & Hatano, 1998a, b) (CB) agar (5 g peptone, 5 g glucose, 3 g yeast extract, 10 g casein peptone, 5 g NaCl, 15 g agar, 1 litre distilled water, and members of the genus Frigoribacterium (Ka$ mpfer pH 7n2–7n4) and incubated for 3 weeks at room temperature et al., 2000) possess the diamino acid lysine in their (18–24 mC). The developed colonies were selected, trans- peptidoglycan. The peptidoglycan of the genus ferred on the same medium, and stored at 8 mC or freeze- Frigoribacterium is characterized by a tetrapeptide dried. composed of glycine, glutamate, homoserine and alanine, and its interpeptide bridge consists of -lysine. Morphology, physiology and cell chemistry. Morphology and life cycle were studied in cultures grown on CB agar In the course of studying micro-organisms from the by phase-contrast and electron microscopy. Physiological plant phyllosphere, we isolated new strains of coryne- features were examined as described previously (Evtushenko form bacteria which were similar to Frigoribacterium et al., 2000). Oxidase activity was tested in a 2-day-old culture using 1% (w\v) tetramethyl-p-phenylenediamine ...... solution on filter paper discs. For the chemotaxonomic The DDBJ accession numbers for the 16S rDNA sequences of Okibacterium study, cultures were grown in liquid CB medium with fritillariae VKM Ac-2059T and Okibacterium fritillariae VKM Ac-2062 are shaking for 20 h. Compositions of cell walls, menaquinones, AB042094 and AB042097, respectively. phospholipids and mycolic acids were studied as given in

02033 # 2002 IUMS Printed in Great Britain 987 L. I. Evtushenko and others

Evtushenko et al. (1989, 2000), and the acyl type of separate branch within the phylogenetic cluster of the peptidoglycan and cellular fatty acids were determined as family Microbacteriaceae, attaching to the cluster of described by Takeuchi & Hatano (1998a). the genus Microbacterium with a 65% bootstrap DNA characterization and hybridization. Genomic DNA was replication value (Fig. 1). Other phylogenetic analyses, isolated from cells resuspended in TE buffer (10 mM Tris, using different sets of strains of the family Micro- 1 mM EDTA; pH 8n0) by the technique described by bacteriaceae (not presented), demonstrated that our Wolfrum et al. (1986). The GjC content was determined by isolates had varying positions within the cluster(s) thermal denaturation as described previously (Evtushenko $ composed of the genera Agreia, Agrococcus, Leifsonia et al., 1989). H -labelled DNAs were obtained using deoxy $ and sometimes Leucobacter with relatively low boot- [1h,2h,5h- H]CTP and the nick translation kit N 5500 strap values – up to 57%. Strains VKM Ac-2059T and (Amersham). DNA–DNA relatedness was studied by the VKM Ac-2062 exhibited the highest 16S rDNA membrane filter method using nylon-6 membranes (0n2 µm; ‘Hiiu Kalur’, Tallinn, Estonia). The hybridization was sequence similarity with Agreia bicolorata (96n6 and performed under optimal conditions (Denhardt’s solution 96n8%, respectively), several members of the genus with 50%, v\v, formamide; 50 mC, 24 h) as described by Microbacterium (Microbacterium testaceum is the Tijssen (1993). closest, 96n6 and 96n8%) and Leifsonia poae (96n3 and 96 6%). However, our strains showed only 94 7– 16S rDNA sequence determination and phylogenetic analy- n n sis. 16S-rRNA-specific DNA was amplified by PCR and 95n5% 16S rDNA sequence similarity with the strains sequenced directly (Hiraishi et al., 1994) with a Thermo of Frigoribacterium faeni, which have similar peptido- Sequenase fluorescent labelled primer cycle sequencing kit glycan amino acids to our strains (see below). with 7-deaza-dGTP (Amersham Pharmacia Biotech), and analysed with a LI-COR 4200 (2-dye system) DNA sequencer following the manufacturers’ protocols. Nucleo- Cell chemistry tide substitution rates were calculated as described by T Kimura & Ohta (1972), and the phylogenetic tree was The cell wall peptidoglycan of strains VKM Ac-2059 constructed by the neighbour-joining method (Saitou & Nei, and VKM Ac-2062 contained lysine, alanine, glycine, 1987) with   software (Thompson et al., 1994). glutamate and homoserine in molar ratios 1n0:1n0:1n1: Tree topologies were evaluated by bootstrap analysis of the 1n1:0n51 and 1n0:1n02:1n02:0n90:0n40, respectively, as sequence data with the same software. well as muramic acid and glucosamine. The reduced content of homoserine was due to formation of lactone RESULTS AND DISCUSSION from some of the homoserine in the hydrolysates. The data on amino acid analyses, along with the sup- Isolation position that the peptidoglycan of our strains is rather of the B-group, like that in all the known members of Several bacterial and fungal colonies were developed the family Microbacteriaceae, and that a diamino acid from the ground seed suspensions on CB agar. Similar is always present in the interpeptide bridge of this coryneform organisms with yellow colonies were peptidoglycan type (Schleifer & Seidl, 1985) provide selected, and the strains VKM Ac-2059T and VKM evidence that lysine residues are positioned at inter- Ac-2062 from seeds of Fritillaria ruthenica and Clema- peptide bridges and, consequently, the tetrapeptide tis recta, respectively, were studied in detail in this subunits consist of glycine, glutamate, homoserine and work. alanine. Although the configurations of the amino acids were not determined, their composition and Morphology molar ratios are similar to those found in peptido- glycan of Frigoribacterium faeni (Ka$ mpfer et al., 2000). The colonies of both strains were yellow, circular, However, the glycan moiety of the peptidoglycan in convex, opaque, glistening and butyrous on CB agar. our strains contained glycolyl residues, in contrast to Young cells (18–20 h) were non-spore-forming, and the acetylated muramic acid of peptidoglycan in irregular-rod-shaped to ovoid (approx. 0n4–0n6i0n7– Frigoribacterium. 1n5 µm). No marked motility or branching was observed. In older cultures, ovoid to irregular coccoid The cell wall sugars of both our strains were similar cells predominated and occurred singly or in pairs with and included rhamnose as the predominant com- diphtheroid arrangements. The electron microscopic ponent and a minor amount of glucose. Isoprenoid study showed that the cell walls were typically Gram- quinones were the major menaquinone MK-10, a positive, with a nearly homogeneous electron-dense somewhat lower content of MK-11, and minor thick coat (peptidoglycan) around the cytoplasmic amounts of MK-9 and MK-8. The principal phospho- membrane, and a less dense irregular outer layer. lipids were phosphatidylglycerol and diphosphatidyl- glycerol. No mycolic acids were found. The fatty acid 16S rDNA sequence analysis profiles were typical of most genera of the family Microbacteriaceae and consisted mainly of saturated Almost complete sequences of 16S rDNA (1506 anteiso- and iso-methyl branched acids: anteiso-15:0 nucleotide bases) were determined in strains VKM Ac- (48% in VKM Ac-2059T and 54% in VKM Ac-2062), T 2059 and VKM Ac-2062. The strains exhibited 99n6% anteiso-17:0 (23 and 19%) and iso-16:0 (29% and 16S rDNA similarity to each other and formed a 20%) acids. Iso-14:0, iso-15:0 and iso-17:0 as well as

988 International Journal of Systematic and Evolutionary Microbiology 52 Okibacterium gen. nov.

...... Fig. 1. Phylogenetic tree showing the position of the strains Okibacterium fritillariae VKM Ac-2059T and Okibacterium fritillariae VKM Ac-2062 based on 16S rDNA analysis. Nucleotide sequence accession numbers are given in parentheses. Brevibacterium linens DSM 20425T (X77451) served as an outgroup sequence (not presented). Bootstrap values, expressed as a percentage of 1000 replications, are shown at the branching points; only the values above 50% are given. Bar, 1 nucleotide substitution per 100 nucleotides. the non-branched acids were variable (1–3%) or not medium supplemented with 0n1% (w\v) yeast extract detected. No 10-methyl branched or ω-cyclohexyl and 0n1% (w\v) casitone. Adonitol, dextrin, dulcitol, undecanoic acids were identified. meso-erythritol and ribose were not used as a carbon source in the same medium. Growth on -melibiose Physiology and salicin was slow or absent in different experiments. Acids were produced from -arabinose, -glucose, Both studied strains were aerobic, and catalase- and mannose, -rhamnose, sucrose, trehalose and -xy- oxidase-positive. Growth occurred between 7 and lose, but not from -melibiose, melezitose, -rham- 37 mC; the optimum was 24–27 mC. -Arabinose, cello- nose, raffinose, -sorbose or sorbitol. Starch and biose, -fructose, -galactose, -glucose, inositol, inu- aesculin were decomposed; hypoxanthine, tyrosine, lin, lyxose, maltose, -mannitol, -mannose, -rham- xanthine, casein, Tween 40, Tween 60 and Tween 80 nose, salicin, sorbitol, sucrose, trehalose and -xylose were not hydrolysed. H#S was weakly produced. Both were used as carbon sources for growth in a mineral strains tolerated 6% (w\v) NaCl but were sensitive to http://ijs.sgmjournals.org 989 L. I. Evtushenko and others

Table 1. Characteristics that differentiate Okibacterium gen. nov. from other genera of the family Microbacteriaceae ...... Data from Schleifer & Kandler (1972), Collins et al. (1983a, b), Davis et al. (1984), Casida (1986), Komagata & Suzuki (1986), Yokota et al. (1993a, b), Zgurskaya et al. (1993), Takeuchi & Yokota (1994), Groth et al. (1996), Takeuchi et al. (1996), Suzuki et al. (1997), Takeuchi & Hatano (1998a, b), Matsuyama et al. (1999), Schumann et al. (1999), Ka$ mpfer et al. (2000), Ma$ nnisto$ et al. (2000), Behrendt et al. (2001), Evtushenko et al. (2000, 2001), and this study. DAB, 2,4-diaminobutyric acid; Orn, ornithine; Lys, lysine; Hyg, 3-hydroxyglutamate; j, positive; k, negative; d, variable between species; , no data.

Genus Peptidoglycan Major MK* Fatty acid GjC, type† mol% Diamino Hyg Glycine in Acyl type acid cross-linkage

Okibacterium gen. nov. Lys kk‡ Glycolyl MK-10, 11 S, A, I 67 Microbacterium Lys\Orn d j§ Glycolyl MK-11, 12, 13, 14 S, A, I 65–72 Frigoribacterium Lys kk Acetyl MK-9 S, A, I 71n7 Agreia DAB, Orn j  Acetyl MK-10 S, A, I 67n0 Agrococcus DAB k  Acetyl MK-11, 12 S, A, I 74 Agromyces DAB kk Acetyl MK-12 S, A, I 71–76 Clavibacter DAB kk Acetyl MK-9 S, A, I 67–78 Cryobacterium DAB kk  MK-10 S, A, I, (12H) 65 Curtobacterium Orn kk Acetyl MK-9 S, A, I, (H) 68–75 Leifsonia DAB kk  MK-11 S, A, I 66–73 Leucobacter DAB kk Acetyl MK-11 S, A, I 66n2 Rathayibacter DAB kk  MK-10 S, A, I 63–72 Subtercola DAB jk  MK-9, 10 S, A, I 64–68

* Designation of Collins & Jones (1981). † S, straight-chain saturated; A, anteiso-methyl-branched; I, iso-methyl-branched; H, cyclohexyl fatty acid; 12H, 12-methyl- tetradecenoic acid; components given in parentheses are sometimes present. Frigoribacterium strains were reported to contain unsaturated fatty acids when grown at low temperature (! 10 mC). ‡ Inferred on the basis of amino acid composition. § Data are based on 28 of 31 species of the genus Microbacterium. No glycine was found in the interpeptide cross-linkage of Microbacterium keratanilyticum and Microbacterium resistens; no precise data on the peptidoglycan amino acid composition of Microbacterium kitamiense are available.

" " erythromycin (15 µgml− ), levomycetin (30 µgml− ), isolates and representatives of the genera Micro- " " neomycin (30 µgml− ), streptomycin (30 µgml− ) bacterium, Agreia and Leifsonia were higher than those " and tetracycline (30 µgml− ). VKM Ac-2059T was between most genera of the order Actinomycetales, " sensitive to kanamycin (30 µgml− ) and lincomycin these values were approximately in the same rank as or " (15 µgml− ), while VKM Ac-2062 tolerated these only slightly exceeded the 16S rDNA sequence simi- antibiotics. larity levels determined between other close genera of the family Microbacteriaceae (e.g. Clavibacter, Base composition of DNA and DNA–DNA Rathayibacter and Frigoribacterium) or some members reassociation study of related genera, particularly from the families Actino- synnemataceae (Labeda & Kroppenstedt, 2000; Yassin The GjC content of DNA was found to be et al., 1995) or Intrasporangiaceae (Stackebrandt et al., T 66n6 mol% in VKM Ac-2059 and 67n2% in VKM 1997; Prauser et al., 1997). The separate phylogenetic Ac-2062. The DNA–DNA reassociation level between position of strains VKM Ac-2059T and VKM Ac-2062 these strains was 85%, indicating both strains to be is in agreement with their distinctions at the phenetic members of common genomic species (Wayne et al., level. The strains differ from most genera and species 1987). of the family Microbacteriaceae, which possess cell wall peptidoglycan based upon 2,4-diaminobutyric Taxonomic affiliation of strains acid or ornithine, by the presence of lysine in their peptidoglycan and also the glycolyl residue of muramic As shown above, strains VKM Ac-2059T and VKM acid, the major menaquinones MK-10 and MK-11, Ac-2062 isolated from plant seeds formed a distinct fatty acid composition, and the 67 mol% GjC lineage within the phylogenetic cluster composed of content of DNA (Table 1). The major menaquinones the genera of the family Microbacteriaceae. Although MK-10 and MK-11, and lack of glycine in the the 16S rDNA sequence similarity values between our interpeptide bridges, are the common characteristics

990 International Journal of Systematic and Evolutionary Microbiology 52 Okibacterium gen. nov.

Table 2. Salient characteristics differentiating Okibacterium gen. nov., Frigoribacterium and Microbacterium species containing lysine in their peptidoglycan ...... Data from Schleifer & Kandler (1972); Collins et al. (1983a); Yokota et al. (1993a); Takeuchi & Yokota (1994); Takeuchi & Hatano (1998b); Ka$ mpfer et al. (2000); and this study. Hsr, homoserine; Glu, glutamate; Gly, glycine; other designations as in Table 1.

Characteristic Okibacterium Frigoribacterium M. aurum M. dextranolyticum M. arborescens gen. nov M. hominis M. imperiale M. lacticum M. laevaniformans

Motility kjkkj Murein type [Hsr]–Glu– Lys* [-Hsr]– [-Hsr]– [-Lys]– [-Hsr]– -Glu– -Glu(Hyg)–Gly– -Glu(Hyg)–Gly– -Glu(Hyg)–Gly#– -Lys -Lys -Lys -Lys Acyl type Glycolyl Acetyl Glycolyl Glycolyl Glycolyl Galactose in cell wall k  jjj Major MK MK-10, 11 MK-9 MK-11, 12 MK-11, 12 MK-11, 12 GjC, mol% 66n6–67n271n769n268n3–71n271n0–71n2

* Inferred on the basis of amino acid composition. which differentiate our strains from 28 out of 31 with rhamnose, 6d-tallose, fucose, mannose and glu- species of the genus Microbacterium of both lysine- cose in different combinations, whereas the cell wall and ornithine-containing groups which are charac- sugars of our strains are represented mainly by terized by peptidoglycan of B1α,B1β and B2β types rhamnose and a minor amount of glucose. (Schleifer & Kandler, 1972; Collins et al., 1983a, b; On the basis of the above phylogenetic and chemo- Yokota et al., 1993a, b; Takeuchi & Hatano, 1998a, b; taxonomic distinctions of the strains VKM Ac-2059T Schumann et al., 1999; Behrendt et al., 2001). The only and VKM Ac-2062 from other genera and species of exceptions are Microbacterium keratanolyticum and the family Microbacteriaceae, we consider that they Microbacterium resistens, which have B2α-type should be placed in a new genus, Okibacterium gen. peptidoglycan with the interpeptide bridge consisting nov. The high level of DNA–DNA relatedness and the of -ornithine (Yokota et al., 1993b; Funke et al., phenetic similarity between these strains indicate that 1998), and no detailed data are available on the they both belong to one species, according to the ornithine-based peptidoglycan of Microbacterium current species definition (Wayne et al., 1987). We kitamiense (Matsuyama et al., 1999). Besides, in propose the name Okibacterium fritillariae for this new contrast to our strains, many microbacterial species species. have 3-hydroxyglutamate in their peptidoglycan. Salient taxonomic properties which differentiate the Description of Okibacterium gen. nov. strains under study from other members of the family Microbacteriaceae with lysine in their cell walls are Okibacterium (O.ki.bac.tehri.um. N.L. n. Oka, the presented in Table 2. As shown, the menaquinone name of the river; Gr. n. bakterion small rod; N.L. pattern, the acyl type of muramic acid, and the GjC neut. n. Okibacterium a bacterium isolated from plants content of DNA markedly distinguish our strains from occurring near the Oka river). the genus Frigoribacterium (Table 2). The other group Cells are Gram-positive, non-sporulating, irregular with lysine-based peptidoglycan (Microbacterium rods. Mesophilic. Aerobic, catalase- and oxidase- arborescens, Microbacterium aurum, Microbacterium positive. Cell wall peptidoglycan of B-type contains imperiale, Microbacterium dextranolyticum, Micro- lysine as a diamino acid. Muramic acid occurs in the bacterium hominis, Microbacterium lacticum and N-glycolyl form. No mycolic acids. The major mena- Microbacterium laevaniformans) is clearly separated quinones are MK-10 and MK-11, with minor amounts from our strains by glycine-containing interpeptide of MK-9 and MK-8. The principal phospholipids are bridges in their peptidoglycan and the presence of 3- phosphatidylglycerol and diphosphatidylglycerol. hydroxyglutamate instead of glutamate, as well as the Among the fatty acids, anteiso-15:0 and anteiso- major menaquinones MK-11 and MK-12 and the 17:0 are predominant. GjC content of DNA is 68n2–71n2mol%GjC content of DNA (Collins et al., approximately 67 mol%. A wide range of carbon 1983a; Yokota et al., 1993a; Takeuchi & Yokota, sources are used for growth. Forms a separate branch 1994; Takeuchi & Hatano, 1998b) (Table 2). In within the phylogenetic cluster of the family Micro- addition, these lysine-containing Microbacterium bacteriaceae. Salient properties that differentiate the species always have galactose in the cell walls, along new genus from other genera of this family are http://ijs.sgmjournals.org 991 L. I. Evtushenko and others summarized in Tables 1 and 2. The type species is this paper had already been accepted for press (Brennan et Okibacterium fritillariae. al., 2001). Like other Microbacterium spp. shown in Table 2, M. gubbeenense is characterized by the presence of galactose in the cell wall, the glycolyl residue of the peptidoglycan and Description of Okibacterium fritillariae gen. nov., sp. the major menaquinones MK-11 and MK-12. The GjC nov. content ranges from 69 to 72 mol%. No detailed data on the peptidoglycan amino acid composition are available. Okibacterium fritillariae (fri.til.lahri.ae. N.L. gen. n. fritillariae of Fritillaria, generic name of plant, the source of isolation of the type strain of the species). REFERENCES General morphological and chemotaxonomic charac- Behrendt, U., Ulrich, A. & Schumann, P. (2001). Description of teristics are as given above in the genus description. Microbacterium foliorum sp. nov. and Microbacterium phyllosphaerae The colonies are yellow, circular, somewhat convex, sp. nov., isolated from the phyllosphere of grasses and the surface litter after mulching the sward, and reclassification of Aureobacterium glistening, opaque, butyrous. Cells are irregular rods resistens (Funke et al. 1998) as Microbacterium resistens comb. nov. Int (length 0n7–1n5 µm, width 0n4–0n6 µm). In older J Syst Evol Microbiol 51, 1267–1276. cultures, ovoid or irregular coccoid cells predominate Brennan, N. M., Brown, R., Goodfellow, M., Ward, A. C., Beres- and occur singly or in pairs with diphtheroid ford, T. P., Vancanneyt, M., Cogan, T. M. & Fox, P. F. (2001). arrangements. No marked motility or primary Microbacterium gubbeenense sp. nov., from the surface of a smear- branching. Growth occurs between 7 and 37 mC, ripened cheese. Int J Syst Evol Microbiol 51, 1969–1976. optimum is 24–27 mC. A wide range of carbon sources Casida, L. E. (1986). Genus Agromyces Gledhill and Casida 1969c, are used for growth in a mineral medium supplemented 346AL.InBergey’s Manual of Systematic Bacteriology, vol. 2, pp. with 0n1% (w\v) yeast extract and 0n1% (w\v) 1329–1331. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & casitone. Polyols are usually not used as a carbon J. G. Holt. Baltimore: Williams & Wilkins. source in the same medium. Acids are produced from Collins, M. D. & Jones, D. (1981). The distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. -arabinose, -glucose, mannose, -rhamnose, Microbiol Rev 45, 316–354. sucrose, trehalose and -xylose. Slow hydrolytic ac- Collins, M. D., Jones, D. & Kroppenstedt, R. M. (1983a). tivity against many substances, but starch and aesculin Reclassification of Brevibacterium imperiale (Steinhaus) and ‘‘Coryne- are decomposed. H#S is weakly produced. Both strains bacterium laevaniformans’’ (Dias and Bhat) in a redefined genus, tolerated 6% (w\v) NaCl. Generally susceptible to Microbacterium (Orla-Jensen) as Microbacterium laevaniformans nov. antibiotics. The physiology details of the two strains rev., comb. nov. Syst Appl Microbiol 4, 65–78. studied in this work are as given above. Cell wall Collins, M. D., Jones, D., Keddie, R. M., Kroppenstedt, R. M. & peptidoglycan contains lysine, alanine, glycine, Schleifer, K. H. (1983b). Classification of some coryneform bacteria in glutamate and homoserine. Rhamnose is the pre- a new genus Aureobacterium. Syst Appl Microbiol 4, 236–252. dominant sugar of the cell wall. G C content of DNA Davis, M. J., Gillaspie, A. G., Vidaver, A. K. & Harris, R. W. (1984). j Clavibacter is about 67 mol%. The type strain is VKM Ac-2059T : a new genus containing some phytopathogenic coryneform T bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and l IFO 16404 , isolated from seeds of Fritillaria Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ruthenica Wikstr. ratoon stunting disease of sugarcane and Bermudagrass stunting disease. Int J Syst Bacteriol 34, 107–117. While this report was being reviewed, the description Evtushenko, L. I., Akimov, V. N., Dobritsa, S. V. & Taptykova, S. D. of Mycetocola gen. nov. within the family Micro- (1989). A new species of actinomycete, Amycolata alni. Int J Syst bacteriaceae, containing lysine in its peptidoglycan, Bacteriol 39, 72–77. was published (Tsukamoto et al., 2001). Mycetocola Evtushenko, L. I., Dorofeeva, L. V., Subbotin, S. A., Cole, J. R. & spp. significantly differ from our strains phylo- Tiedje, J. M. (2000). Leifsonia poae gen. nov., sp. nov., isolated from genetically (95n4–95n8% of the 16S rDNA sequence nematode galls on Poa annua, and reclassification of ‘Corynebacterium similarity) and phenotypically, mainly by the presence aquaticum’ Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with of an unknown amino acid in peptidoglycan (one two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov. molecule per unit), the molar ratios of glutamate, Int J Syst Evol Microbiol 50, 371–380. glycine, alanine and lysine, and also the lack of Evtushenko, L. I., Dorofeeva, L. V., Dobrovolskaya, T. G., homoserine; the major menaquinone MK-10 and Streshinskaya, G. M., Subbotin, S. A. & Tiedje, J. M. (2001). Agreia minor menaquinones MK-9 and MK-11; a lower bicolorata gen. nov., sp. nov., to accommodate isolated GjC content of DNA (63n9–65n2 mol%); and cell from narrow reed grass infected by the nematode Heteroanguina shape and size (0n2–0n4i2n0–3n5 µm). graminophila. Int J Syst Evol Microbiol 51, 2073–2079. Funke, G., Lawson, P. A., Nolte, F. S., Weiss, N. & Collins, M. D. (1998). Aureobacterium resistens sp. nov., exhibiting vancomycin ACKNOWLEDGEMENTS resistance and teicoplanin susceptibility. FEMS Microbiol Lett 158, 89–93. This work was financially supported by grant no. 00-04- Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. 49074 of the Russian Foundation of Basic Research. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239. NOTE ADDED IN PROOF Hiraishi, A., Shin, Y. K., Ueda, Y. & Sugiyama, K. (1994). 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992 International Journal of Systematic and Evolutionary Microbiology 52 Okibacterium gen. nov.

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