Taxonomic Significance of 2,4=Diaminobutyric Acid Isomers In

International Journal of Systematic Bacteriology (1 998), 40,403-41 0 Printed in Great Britain

Taxonomic significance of 2,4=diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov.

Junko Sasaki,’ Masao Chijimatsu’ and Ken-ichiro Suzuki’

Author for correspondence: Ken-ichiro Suzuki. Tel: +81 48 467 9559. Fax: +81 48 462 4617. e-mail: [email protected]

Japan Collection of An HPLC procedure which separates D- and L-amino acid isomers was applied to Microorganisms1and an analysis of peptidoglycan of 2,4=diaminobutyric acid (DAB)-containing Division of Biomolecular Characterization,* The actinomycetes. The cell wall peptidoglycans of species of the genera Institute of Physical and Agromyces, CIavibacter and Rathayibacter contain DAB and have been Research (RIKEN)r differentiated principally by their menaquinone profile. These peptidoglycans Wa ko-shi, Saitama 351-0198, Japan are known to be identical in structure, all being of the B2y type, possessing both D- and L-DAB. The type strains of all the subspecies of CIavibacter michiganensis have D- and L-DAB in almost equal proportions in their cell wall peptidoglycan as previously reported. In contrast, the type strains of CIavibacter toxicus and all valid species of the genera Agromyces and Rathayibacter contain the L-isomer of DAB almost exclusively. This characteristic is in good agreement with phylogenetic analyses based on 165 rDNA sequences and menaquinone profiles. On the basis of these data, the transfer of CIavibacter toxicus to the genus Rathayibacter as Rathayibacter toxicus comb. nov. is proposed. The isomer profile of DAB is shown to be a good taxonomic marker to differentiate these genera.

I Keywords : Rathayibacter toxicus comb. nov., 2,4-diaminobutyric acid isomers, peptidoglycan, actinomycetes, Clavibacter toxicus

INTRODUCTION diamino acids have been found in group B peptidoglycan. Many variations have been found in the cell wall peptidoglycan of Gram-positive bacteria and the It is difficult to determine peptidoglycan grouping characteristic diamino acids have been employed as simply from amino acid composition. The tetrapeptide useful chemotaxonomic markers (Schleifer & Kandler, subunit of bacterial peptidoglycan contains peptides 1972; Yamada & Komagata, 1972b). Group A pep- composed of both L- and D-amino acids. Therefore, tidoglycan, commonly found in most bacterial cell the ability to distinguish D- and L-amino acid isomers would be of great value in the elucidation of the amino walls, contains an L- or meso-diamino acid at position 3 in the peptide subunit. In contrast, group B peptido- acid composition of peptidoglycan, particularly group glycan has a characteristic diamino acid in the inter- B peptidoglycan. peptide bridge which connects D-glutamic acid at The determination of amino acid isomers by HPLC position 2 and D-alanine at position 4 in peptide has been applied to isomers of diaminopimelic acid in subunits (Fiedler et al., 1970; Fiedler & Kandler, 1973; actinomycete taxonomy (Puchala et al., 1992 ; Taka- Schleifer & Kandler, 1972). Both L- and D-form hashi et al., 1989; Tisdall & Anhalt, 1979). It has been reported that 17 primary amino acid enantiomers can Abbreviations: DAB, 2,Qdiaminobutyric acid; FLEC, (+)-1-(9-fluorenyl)- be separated by reversed-phase HPLC following pre- ethyl chloroformate; GABA, y-aminobutyric acid. column derivatization with ( + )- 1-(9-fluorenyl)ethyl The DDBJ accession numbers for the sequences reported in this paper are chloroformate (FLEC) (Einarsson & Josefsson, 1987; listed in Table 1. Hayashi & Sasagawa, 1993). As the peptidoglycan of

00591 Q 1998lUMS 403 J. Sasaki, M. Chijimatsu and K.4. Suzuki

Table 7. Strains used in this study and accession numbers of their 165 rDNA sequences

Taxon JCM no.* Other designationt Source (reference) Accession no. (reference)

~~~ ~

Agrococcus jenensis 9950' DSM 9850T Frozen compost soil (Groth et al., 1996) X92492 (Groth et al., 1996) Agrococcus jenensis 995 1 DSM 9996 Agromyces cerinus subsp. cerinus 9083' VKM Ac- 1340T Soil (Zgurskaya et a[., 1992) D45060 (Suzuki et al., 1996) Agromyces cerinus subsp. nitratus 9084T VKM Ac-1351T Soil (Zgurskaya et al., 1992) Agromyces fucosus subsp. fucosus 9085T VKM Ac-1345' Soil (Zgurskaya et al., 1992) Agromyces fucosus subsp. hippuraius 9086' VKM Ac-1352T Soil (Zgurskaya ei al., 1992) D45061 (Suzuki ei al., 1996) Agromyces mediolanus 3346T DSM 20152T (Mamoii, 1939) X77449 (Rainey et al., 1994) Agromyces ramosus 3108' DSM 43045T Soil (Gledhill & Casida, 1969) X77447 (Rainey et al., 1994) ' Brevibacteriwn helvolm' 949 1 DSM 20419 Butter X77440 (Rainey er al., 1994) Clavibacter michiganensis subsp. insidiosus 1369 ATCC 10253 D45051 (Suzuki et a/., 1996) Clavibacier michiganensis subsp. insidiosus 9664' ICMP 2621T Lucerne Clavibacier michiganensis subsp. michiganensis 9665' DSM 463UT Tomato X77435 (Rainey ei al., 1994) Clavibacter michiganensis subsp. michiganensis 1370 ATCC 492 Tomato D84128 (this study) Clavibacier michiganensis subsp. nebraskensis 9666' DSM 7483T Maize leaf freckle X77434 (Rainey et al., 1994) Clavibacier michiganensis subsp. sepedonicus 9667' ICMP 2535' Solanum tuberosum Clavibacter michiganensis subsp. tessellarius 9668= ATCC 33566T Winter wheat U30254 Clavibacter toxicus 9669T ICMP 9525T Ryegrass (Riley & Ophel, 1992) D84127 (this study) Clavibacter xyli subsp. cynodontis 9733' ICMP 8790' Bermudagrass (Davis ei al., 1984) ' Corynebacierium aquaticum' 1368 ATCC 14465 Distilled water (Leifson, 1962) D45057 (Suzuki et al., 1996) Cryobacterium psychrophilum 1463T IAM 12024T Antarctic soil (Inoue BE Komagata, 1976) D45058 (Suzuki et al., 1997) L.eucobacter komagatae 94 14' IF0 15245T (Takeuchi et al., 1996) D17751 (Takeuchi et al., 1996) Rathayibacier iranicus 93OgT ICMP 3496T Triticum aestivum Raihayibacier raihayi 9307' DSM 748F Dactylis glomerata X77439 (Rainey ei al., 1994) Rathayibacter iritici 9309' DSM 7486T Triticwn aestivwn X77438 (Rainey et a/., 1994) * JCM, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Wako, Japan. t ATCC, American Type Culture Collection, Rockville, MD, USA; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; IAM, IAM Culture Collection, Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan ; ICMP, International Collection of Microorganisms from Plants, Manaaki Whenua Landcare Research, Auckland, New Zealand ;NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, UK ; VKM, All-Russian Collection of Microorganisms, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia. bacterial cell walls contains several unusual amino This study deals with the taxonomic significance of D- acids such as ornithine and 2,4-diaminobutyric acid and L-DAB isomers in actinomycetes. Based on the (DAB), we have tried to apply the technique, with data collected, together with other chemotaxonomic some modifications, to bacterial peptidoglycan analy- and phylogenetic data, we discuss the delineation of sis. the DAB-containing genera and propose the transfer The actinomycetes, with peptidoglycan containing of Clavibacter toxicus to the genus Rathayibacter as DAB, comprise six genera: Agrococcus (Groth et al., Rathayibacter toxicus comb. nov. 1996), Agromyces (Fiedler & Kandler, 1973; Gledhill & Casida, 1969; Suzuki et al., 1996; Zgurskaya et al., METHODS 1992), Clavibacter (Davis et al., 1984; Riley & Ophel, Bacterial strains and cultivation. Actinomycetes containing 1992), Cryobacterium (Suzuki et al., 1997), Leuco- DAB in the cell wall were used in this study and are listed in bacter (Takeuchi et al., 1996) and Rathayibacter Table 1. All the type strains of valid species of the genera (Collins, 1983; Zgurskaya et al., 1993). The genera Agromyces, Clavibacter and Rathayibacter, except that of Agromyces, Clavibacter and Rathayibacter share a Clavibacter xyli subsp. xyli, were included. Strains of the common peptidoglycan structure of the B2y type as recently proposed genera Agrococcus, Cryobacterium and described by Schleifer & Kandler (1972) and are Leucobacter were also studied. differentiated mainly by their menaquinone profiles Biomass was collected from bacteria cultivated at 30 "C for (Collins, 1982, 1983; Suzuki et al., 1996; Zgurskaya et 48 h on R medium on a rotary shaker unless otherwise stated al., 1992, 1993). B2y-type peptidoglycans have also (Suzuki et al., 1996). R medium contains (1-l) log Bacto been found in ' Corynebacteriumaquaticum' JCM 1368 Peptone (Difco), 5 g yeast extract (Difco), 5 g malt extract and 'Brevibacterium helvolum ' JCM 9491, though there (Difco), 5 g Casamino acids (Difco), 2 g beef extract (Difco), is no suitable taxa to accommodate these invalid 2 g glycerol, 50 mg Tween 80 and 1 g MgSO, .7H,O, pH 7.2 species. Several other peptidoglycan structures con- (Yamada & Komagata, 1972a). Cryobacterium psychro- taining DAB have been reported in some Gram- philum JCM 1463Twas cultivated at 10 "C for 5 d. Clavi- bacter xyli subsp. cynodontis JCM 9733T was cultivated at positive bacteria (Bendinger et al., 1992; Fiedler & 28 "C for 5 d on SC medium. SC medium contains (1-l) 17 g Kandler, 1973; Groth et al., 1996; Hensel, 1984; Cornmeal agar (Difco), 8 g Phytone Peptone (Difco), 1 g Takeuchi et al., 1996). However, no taxa have been KH,PO,, 1 g K,HPO,, 0.2 g MgSO,. 7H,O, 2 g bovine established for these strains except Agrococcusjenensis serum albumin, 0-5 g glucose, 1 g cysteine free base and (Groth et al., 1996) and Leucobacter komagatae 15 mg bovine haemin chloride (Sigma), pH 6.6 (Davis et al., (Takeuchi et al., 1996) which were proposed recently. 1984).

404 International Journal of Systematic Bacteriology 48 Rathayibacter toxicus comb. nov.

Table 2. Amino acid profiles of cell wall peptidoglycan from DAB-containing actinomycetes

Strain Molar ratio to Glu* Isomer of DAB DIL (%I Mur Glc-NH, Glu GlY Ma DAB Asp GABA Thr

Agromyees A. cerinus subsp. cerinus JCM 9083T 033 0.38 1.oo 1.oo 0.67 1.91 0 0 0 4/96 A. cerinus subsp. nitratus JCM 9084' 043 0.49 1.oo 0.97 0.67 1.71 0 0 0 2/98 A. fucosus subsp.fucosus JCM 9085T 0.34 0.49 1.oo 1 .oo 0.73 1.72 0 0 0 2/98 A. fucosus subsp. hippuratus JCM 9086T 034 0.40 1.00 1.02 0.72 1.89 0 0 0 2/98 A. mediolanus JCM 3346T 0.35 0.59 1 .oo 1.01 071 1.65 0 0 0 3/97 A. ramosus JCM 3108* 0.43 0.71 I .oo 1.02 0.63 1.58 0 0 0 4/96 Clavibacter C. michiganensis subsp. insidiosus JCM 9664' 038 0.4 1 1.oo 1.05 0.93 1.80 0 0 0 50/50 C. michiganensis subsp. michigunensis JCM 9665' 045 0.5 1 1 .oo 1.01 0.82 1.41 0 0 0 56/44 C. michiganensissubsp. michiganensis JCM 1370 043 0.70 1.oo 1.03 0.80 1.42 0 0 0 59/41 C. michiganensissubsp. nebraskensis JCM 9666T 033 0-41 1 .oo 1.03 0.73 1.60 0 0 0 54/46 C. michiganensis subsp. sepedonicus JCM 9667' 033 0.38 1.oo 1.07 0.97 1.55 0 0 0 52/48 C. michiganensissubsp. tesselluriusJCM 9668T 038 0.62 1.oo 1-07 0.83 1.59 0 0 0 60140 C. xyli subsp. cynodontis JCM 9733T 0.37 0.84 1.00 0.94 0.76 1.19 0 0 0 62/38 C. toxicus JCM 9669T 0.47 0.43 1.oo 0.95 0.90 1.18 0 0 0 13/87 Rathayibacter R. iranicus JCM 9308= 023 0-31 1.oo 1.04 0.78 1.75 0 0 0 2/98 R. rathayi JCM 9307T 0.3 1 0.37 1.oo 1.06 0.79 1.81 0 0 0 2/98 R. tritici JCM 9309' 0.37 0.50 1.oo 1.08 0.72 1.90 0 0 0 10190 Others Agrococnts jenensis JCM 9950T 0.67 0.70 1 .oo 1.86 2.76 0.96 0.93 0 0.9 1 7/93 Agrococcusjenensis JCM 9951 0.5 1 0.55 1.oo 1.45 2.08 0.67 0.73 0 0.67 10190 ' Brevibacterium helvolum ' JCM 949 1 0.22 0.57 1 .oo 0.98 0.76 1.76 0 0 0 46/54 ' Corynebacterium aquuricum' JCM 1368 0.28 0.47 1 .oo 194 066 1.66 0 0 0 58/42 Cryobacteriumpsychrophilum JCM 1463 0.30 030 1 .oo 0.96 0.60 1.31 0 0 0 16/84 Leucobacter komagatae JCM 9414T 0.43 0.65 1.00 0.75 1.62 0.75 0 0.32 0 4/96 * Mur, muramic acid; Glc-NH,, glucosamine.

with 1 m16 M HCl for 16 h. The hydrolysate was filtered and Glyi n concentrated to dryness by a rotary evaporator. The residue was dissolved in 1 ml water and dried twice to remove HC1. The residue was finally dissolved in 300 pl water and used for amino acid analysis. Amino acid analysis. Amino acid compositions were determined using an automatic amino acid analyser (Hitachi model L-8 500A) equipped with an ion-exchange column (Hitachi #2620MSC). Analysis of amino acid isomers was carried out using HPLC according to the method of Einarsson & Josefsson (1987) with some modifications. L-DAB Amino acids were derivatized with FLEC to fluorescent amino acid diastereomers using the precolumn method and separated by a reversed-phase column (Pegasil ODs-3, 6 x 250 mm; Senshu Scientific). The column was eluted with a programmed gradient of 0.1 M sodium acetate buffer (pH 4.1 7)/acetonitrile/tetrahydrofuran (76 : 12 : 12, by vol.) to 0.1 M sodium acetate buffer (pH 4-46)/acetonitrile/ tetrahydrofuran (4:3: 3, by vol.). The excitation and emission wavelengths for the fluorescence detector were 265 and 315 nm respectively. lsoprenoid quinones. Isoprenoid quinone compositions were 0 50 100 150 determined by the method described previously (Komagata Time (min) & Suzuki, 1987). DNA studies. DNA was extracted by the phenol method (Saito & Miura, 1963). DNA base composition was de- Fig, 1. HPLC chromatogram of FLEC derivatives of amino acid stereoisomers from the peptidoglycan hydrolysate of (a) termined using HPLC (Tamaoka & Komagata, 1984). Clavibacter michiganensis subsp. michiganensis JCM 1370 and Levels of DNA relatedness were determined by the method (b) Agromyces mediolanus JCM 3346T. of Ezaki et al. (1989) using photobiotin and micro- immunoplates. 165 rDNA sequence analysis. 16s rRNA gene sequencing was Preparation of cell wall peptidoglycan. Cell wall peptido- carried out by the method described previously (Suzuki et glycan was prepared according to the method described al., 1996). The sequences determined in this study have been previously (Komagata & Suzuki, 1987). One or two milli- deposited in the DDBJ data library under the accession grams of peptidoglycan sample was hydrolysed at 100 "C numbers shown in Table 1. The phylogenetic tree was

~~~ ~ In terna tiona I lo urnaI of Systematic Bacteriology 48 405 J. Sasaki, M. Chijimatsu and K.4. Suzuki created by the neighbour-joining method using K,,, values same molar ratio of glutamic acid to alanine to glycine (Saitou & Nei, 1987). The topology of the tree was evaluated to DAB [1:(0~6-0~9):(0~9-1~1):(1*2-1~9)]. by performing a bootstrap analysis using the CLUSTAL w program (Thompson et al., 1994). Analysis of amino acid stereoisomers Nucleotide sequence accession numbers. The 16s rDNA sequences listed in Table 1 were aligned with the following The FLEC derivatization technique made it possible published sequences from DDBJ, GenBank and EMBL: to separate and identify chiral amino acids, including Aureobacterium barkeri DSM 20145T 0(77446), Aureo- lysine, ornithine, diaminopimelicacid and DAB. Table bacterium testaceum DSM 20166T 0(77445), Clavibacter xyli 2 shows the ratio of D- to L-isomers of DAB in the subsp. cynodontis MDEl (M60935), Curtobacterium citreum peptidoglycan of the strains studied. Among the strains DSM 20528T(X77436), Curtobacterium luteum DSM 20542T studied, except those of Agrococcus and Leucobacter, (X77437), Microbacterium imperiale DSM 20530T(X77442) two types of amino acid composition were found as and MicrobacteriumlacticumDSM 20427=0(77441).Strepto- shown in Fig. 1. One type contained an almost equal myces griseus KCTC 9080T (X55435) was used as an outgroup member. mix of L- and D-DAB in approximately equal proportions. In the other type the L-isomer was dominant. RESULTS The alanine and glutamic acid residues from the peptidoglycan of the strains tested were exclusively D- Amino acid composition of peptidoglycan isomers. The molar ratios of amino acids to glutamic acid in the peptidoglycan hydrolysate of DAB-containing strains Menaquinone analysis are shown in Table 2. All the strains studied, except for The menaquinone composition of the strains studied is those of Agrococcus and Leucobacter, had almost the shown in Table 3.

Table 3. Menaquinone composition of DAB-containing actinomycetes

Strain Menaquinone composition (YO)*

~~ MK-7 MK-8 MK-9 MK-10 MK-11 MK-12 MK-13 MK-14

Agromyces A. cerinus subsp. cerinus JCM 9083T TR 1 7 8 60 21 1 A. cerinus subsp. nitratus JCM 9084T 1 TR 5 7 61 24 2 A. fucosus subsp. fucosus JCM 9085T 1 6 9 67 15 1 A. fucosus subsp. hippuratus JCM 9086T TR 1 7 9 57 24 2 A. mediolanus JCM 3346T TR 2 1 14 18 59 4 A. ramoms JCM 310ST 1 1 4 8 8 62 15 1 Clavibactev C. michiganensis subsp. insidiosus JCM 9664T 2 4 82 9 C. michiganensis subsp. michiganensis JCM 9665T TR 4 78 13 C. michiganensis subsp. michiganensis JCM 1370 6 4 45 40 5 C. michiganensis subsp. nebraskensis JCM 9666T 7 1 85 7 C. michiganensis subsp. sepedonicus JCM 9667T 1 2 67 25 2 C. michiganensis subsp. tessellarius JCM 9668T 1 4 83 11 C. toxicus JCM 9669T TR 8 73 18 C. xyli subsp. cynodontis JCM 9733T TR 17 44 39 Rathayibacter R. iranicus JCM 9308T TR 18 75 TR R. rathayi JCM 9307T TR 15 74 6 R. tritici JCM 9309T 10 83 7 Others Agrococcus jenensis JCM 9950Tt 7 37 49 5 Agrococcus jenensis JCM 995 1t 4 41 41 13 ' Brevibacterium helvolum ' JCM 9491 TR 4 88 4 ' Corynebacterium aquaticum' JCM 1368 1 9 14 32 35 8 TR Cryobacteriumpsychrophilum JCM 1463$ 3 14 11 50 17 2 Leucobacter komagatae JCM 9414T 2 10 8 73 4

* TR, Trace. ?Data from Groth et al. (1996). $ Data from Suzuki et al. (1997).

406 International Journal of Systematic Bacteriology 48 Rathayibacter toxicus comb. nov.

Leucobacter komagatae JCM 9414' Clavibacter xyli subsp. cynodonfis MDE1 "Corynebacterium aquaticum" JCM 1368 Agromyces mediolanus DSM 201 52 Agromyces ramosus DSM 43045 Agromyces fucosus subsp. hippurafus JCM 9086 Agromyces Agromyces cerinus subsp. cerinus JCM 9083 Ciavibacter toxicus JCM 9669' 100 Rathayibacter rathayi DSM 7485T IRathayibacter -Rathayibacter tritici DSM 7486 ' 1 Cutta6acterium citreum DSM 20528T Curtobacterium Curtobacterium lufeum DSM 20542T 1 1"Brevibacteriurnhelvolum" DSM 20419 Clavibacter rnichiganensis subsp. rnichiganensis DSM 46364T Clavibacter rnichiganensis subsp. michiganensis JCM 1 370 1 LClavibacter rnichiganensis subsp. insidiosus JCM 1369 Clavibacter Clavibacter rnichiganensis subsp. fessellarius ATCC 33566 Clavibacter rnichiganensis subsp. nebraskensis DSM 7483T Cryobacterium psychrophilum JCM 1463T Agrococcus jenensis DSM 9850' I I Aureobacterium barkeri DSM 201 45' 1 11 00 Microbacterium lacticurn DSM 20427T Microbacterium 89 A ureobacterium Microbacterium imperiale DSM 20530 LAureobacteriumtestaceurn DSM 201 66' J I Strepfomyces griseus KCTC 9080T 0.01 t I Knuc

Fig. 2. Phylogenetic tree derived from 165 rDNA sequences of actinomycetes containing DAB in their cell wall. The tree was created by the neighbour-joining method and Knucvalues. The numbers on the tree indicate bootstrap values for the branch points. The strains used and the nucleotide sequence accession numbers are indicated in Table 1.

Table 4. DNA base composition and levels of DNA-DNA relatedness between Clavibacter toxicus JCM 966gT and Ra thayibacter sp p.

Strain G + C content (mol%) Percentage DNA-DNA reassociation with :

JCM 9669T JCM 930F JCM 9308T JCM 9WT JCM 1370

Clavibacter toxicus JCM 9669T 60 100 19 20 22 19 Rathayibacter rathayi JCM 9307T 67 15 100 27 50 15 Rathayibacter iranicus JCM 930gT 66 19 32 100 36 19 Rathayibacter tritici JCM 930gT 69 21 59 38 100 21 Clavibacter michiganensis subsp. 73 17 22 20 23 100 michiganensis JCM 1370

Phylogenetic analysis subsp. cynodontis JCM 9733T were not included in the cluster of Clavibacter michiganensis subspecies. Clavi- A phylogenetic tree of actinomycetes with group B bacter toxicus JCM 966gT was closely associated with peptidoglycan based on 16s rDNA sequences is shown a cluster that contained Rathayibacter species at a in Fig. 2. The phylogenetic tree shows that Agromyces 96 % bootstrap confidence level. CZavibacter xyli species and the group of C. michiganensis subspecies subsp. cynodontis JCM 9733T formed a cluster with form independent branches at high bootstrap levels. ' Corynebacterium aquaticum ' JCM 1368 positioned Clavibacter toxicus JCM 966gT and Clavibacter xyli close to the Agromyces cluster. Agrococcus jenensis international Journal of Systematic Bacteriology 48 407 J. Sasaki, M. Chijimatsu and K.-i. Suzuki

JCM 9950T, Cryobacterium psychrophilum JCM 1463T aquaticum ' JCM 1368 and ' Brevibacterium helvolum ' and Leucobacter komagatae JCM 9414T formed a JCM 9491 also had this type of peptidoglycan. This separate branch from all other clusters. composition is in accordance with the B2y-type structure proposed by Schleifer & Kandler (1972) as shown DNA base composition and DNA relatedness in Fig. 3. On the other hand, the L-isomer was found almost exclusively in the cell wall of all the type strains As Clavibacter toxicus JCM 9669T formed a phylo- of Agromyces and Rathayibacter, and in Clavibacter genetic cluster with Rathayibacter species, the DNA toxicus JCM 9669T and Cryobacterium psychrophilum relatedness of Clavibacter toxicus JCM 9669T to the JCM 1463T.This result suggests that these organisms type strains of Rathayibacter species was examined. have a different peptidoglycan structure to the B2y The DNA base composition of Clavibacter toxicus type. Considering the amino acid composition, it may JCM 9669Twas 60 mol % G + C,whereas that of three be that D-DAB in the interpeptide bridge might be type strains of Rathayibacter species ranged from 66 to substituted by L-DAB in the latter type. It is true that 69 mol %. The relative levels of DNA-DNA reassoci- the isomer profile of DAB clearly differentiates the ation between Clavibacter toxicus JCM 9669T and the strains of the genus Clavibacter from those of Rathayi- three type strains of Rathayibacter species were less bacter and Agromyces, though the structure has not than 22 % as shown in Table 4. The levels of similarity been completely determined. between Rathuyibacter iranicus JCM 930gT, Rathayi- bacter rathayi JCM 9307T and Rathayibacter tritici The menaquinone profile has been one of the most JCM 9309T ranged from 27 to 59%. useful taxonomic markers for DAB-containing actinomycetes (Collins & Jones, 1980; Davis et al., 1984; DISCUSSION Zgurskaya et al., 1992, 1993) and shows a good correlation with the phylogenetic analysis based on Amino acids in peptidoglycan have been widely ac- 16s rDNA sequences (Rainey et al., 1994; Suzulu et cepted as a useful chemotaxonomic marker for Gram- al., 1996). In this study, we have found that the positive bacteria. Analysis of the isomers of amino phylogenetic clusters are homogeneous with respect to acids has revealed further variation of peptidoglycan their peptidoglycan DAB isomer type. Determination in the DAB-containing actinomycetes. Except for of DAB isomers in combination with menaquinone strains of the genera Agrococcus and Leucobacter, the composition enables us to clearly distinguish Clavi- molar ratio of glutamic acid to alanine to glycine to bacter, Rathayibacter and Agromyces. DAB in the cell walls of the strains tested could be summarized as 1: 1 : 1: 2. This result is in accordance with that of B2y-type peptidoglycan (Schleifer & The genus CIavibacter Kandler, 1972). However, among these strains, two patterns of DAB isomer composition were found in All the strains of the subspecies of Clavibacter mich- the cell wall. One type contained almost equal amounts iganensis are characterized by the presence of both D- and L-DAB in their peptidoglycan and MK-9 as the of both D- and L-DAB, while the other contained mostly the L-isomer. predominant menaquinone. These results are sup- ported by high values of DNA relatedness among Peptidoglycan containing almost equal amounts of these subspecies (Dopfer et al., 1982; Zgurskaya et al., both D- and L-DAB was found in the cells of all the 1993) and the phylogenetic analysis derived from 16s strains belonging to the genus Clavibacter except rDNA sequences (Fig. 2). Clavibacter michiganensis Clavibacter toxicus JCM 9669T. ' Corynebacterium subsp. michiganensis JCM 1370 contained almost equal proportions of MK-9 and MK-10 and seemed to be intermediate between the genera Clavibacter and Rathayibacter from the viewpoint of the menaquinone profile. However, the DAB isomer composition of cell wall peptidoglycan indicated that strain JCM 1370 clearly belongs to the genus Clavibacter as the phylogenetic position indicated. While Clavibacter xyli subsp. cynodontis JCM 9733T i J.- if has both D- and L-DAB in its peptidoglycan, the & D-Ala D-GIu-+ predominant menaquinones are MK- 10 and MK- 11. These chemotaxonomic characteristics are the same as those of ' Corynebacteriurn aquaticum ' JCM 1368 and these two taxa form an independent cluster from the genus Clavibacter in the group B actinomycetes. These results suggest that a new genus should be established to accommodate these two strains. Fig. 3. Fragment of the primary structure of B2y type peptidoglycan (Schleifer 81 Kandler, 1972). MurNAc, A/- Clavibacter toxicus JCM 9669Tpeptidoglycan contains acetylmura mic acid; GlcNAc, N-acetylg1 ucosa mine. L-DAB almost exclusively and MK-10 is the pre-

408 International Journal of Systematic Bacteriology 48 Rathayibacter toxicus comb. nov. dominant menaquinone. These characteristics are the Leucobacter komagatae JCM 9414T also has a unique same as those of the genus Rathayibacter. Based on cell wall amino acid composition. One mole each of D- these data and the phylogenetic analysis, this organism and L-alanine, L-DAB, y-aminobutyric acid (GABA), should be transferred to the genus Rathayibacter. The glycine and D-glutamic acid are contained in a unit of levels of DNA relatedness between Clavibacter toxicus peptidoglycan. The amino acid composition of this JCM 9669T and the three type strains of known organism is characterized by the presence of GABA Rathayibacter species indicate that Clavibacter toxicus and D-alanine in the peptidoglycan. is an independent species in the genus Rathayibacter (Table 4). We propose the reclassification of Clavi- ' Brevibacterium helvolum ' JCM 949 1 contains both D- bacter toxicus as Rathayibacter toxicus comb. nov. as and L-DAB in the cell wall peptidoglycan and the described below. Zgurskaya et al. (1993) have already presence of MK-9 puts it in the same group as suggested this from the viewpoint of phenotypic Clavibacter michiganensis subspecies. However, characteristics. However, they did not transfer the 'Brevibacterium helvolum' JCM 9491 stands at a species because they did not include the type strain in distinct position from the Clavibacter michiganensis their study. Further description and comparison with subspecies cluster in the phylogenetic tree. other Rathayibacter species are reported by Riley & Ophel (1992) and Zgurskaya et al. (1993). Description of Rathayibacter toxicus (Riley and Ophel Considering the results mentioned above, it is reason- 1992) comb. nov. able that the genus Clavibacter should be restricted to Rathayibacter toxicus (basonym Clavibacter toxicus the organisms which have L- and D-DAB in almost Riley and Ophel 1992). Cells are Gram-positive, equal proportions in their peptidoglycan and MK-9 as obligately aerobic and non-spore-forming rods (gen- the predominant menaquinone, i.e. subspecies of erally 0.6475 x 1-5pm). Colonies on R agar are flat, Clavibacter michiganensis. smooth, pale yellow after 5 d culture at 30 "C. No growth at 37 "C. The molar ratio of amino acids D- The genus Rathayibacter glutamic acid to glycine to D-alanine to L-DAB in the cell wall peptidoglycan is 1 : 1 : 1 : 2. The predominant The genus Rathayibacter is characterized by possessing isoprenoid quinone is MK- 10. Acid is produced from L-DAB-type peptidoglycan and MK- 10 as the pre- galactose, mannose and xylose. Acetate, glutamate, dominant menaquinone. malonate, succinate and tartrate are not utilized. Positive for H,S production from cysteine. Negative The genus Agromyces for hydrolysis of starch and gelatin. The type strain is JCM 9669 (= ICMP 9525 = NCPPB 3552), isolated The cell walls of the four species and two subspecies of from ryegrass in South Australia. Toxicity to grazing Agromyces contain mostly L-DAB. This genus is animals was observed. The G+C content of the type homogeneous in this characteristic. Although the strain is 60.4 mol YO. predominant menaquinone is MK- 12, the second component is different in the genus. The Agromyces strains are placed in one cluster with a high bootstrap ACKNOWLEDGEMENTS confidence level in the phylogenetic tree. We thank K. Komagata for a critical review of the manuscript. The others REFERENCES Cryobacterium psychrophilum JCM 1463T is a dis- tinctive organism based on its obligately psychrophilic Bendinger, B., Kroppenstedt, R. M., Klatte, 5. & Altendorf, K. growth and cellular fatty acid composition, and forms (1 992). Chemotaxonomic differentiation of coryneform bacteria an independent branch in the phylogenetic tree ac- isolated from biofilters. Int J Syst Bacteriol42, 474486. cording to 16s rDNA sequence data (Suzuki et al., Collins, M. D. (1982). Lipid composition of Agromyces ramosus 1997). This genus is characterized by L-DAB-type (Gledhill and Casida). FEMS Microbiol Lett 14, 187-189. peptidoglycan. Collins, M. D. (1983). Cell wall peptidoglycan and lipid composition of the phytopathogen Corynebacterium rathayi Agrococcus jenensis strains have a unique cell wall (Smith). Syst Appl Microbiol4, 193-198. amino acid composition (Table 2) and are allocated to Collins, M. D. &Jones, D. (1980). Lipids in the classification and an independent position on the phylogenetic tree (Fig. identification of coryneform bacteria containing peptidoglycans 2). The molar ratio of amino acids in the peptidoglycan based on 2,4-diaminobutyric acid. J Appl Bacteriol48,459-470. was in accordance with the data reported by Groth et Davis, M. J., Gillaspie, A. G., Jr, Vidaver, A. K. & Harris, R. W. al. (1996). The major menaquinones of the genus (1984). Clavibacter: a new genus containing some phyto- Agrococcus are MK-11 and MK-12. Peptidoglycan pathogenic coryneform bacteria, including Clavibacter xyli with a similar amino acid composition has been subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. reported in some bacteria isolated from biofilters cynodontis subsp. nov., pathogens that cause ratoon stunting (Bendinger et al., 1992) for which no taxa have been disease of sugarcane and Bermudagrass stunting disease. Int J proposed. Syst Bacteriol34, 107-1 17.

International Journal of Systematic Bacteriology 48 409 J. Sasaki, M. Chijimatsu and K.-i. Suzuki

DtJpfer, H., Stackebrandt, E. & Fiedler, F. (1982). Nucleic acid Saito, H. & Miura, K. (1963). Preparation of transforming hybridization studies on Microbacterium, Curtobacterium, deoxyribonucleic acid by phenol treatment. Biochim Biophys Agromyces and related taxa. J Gen Microbioll28, 1697-1708. Acta 72, 619-629. Einarsson, 5. & Josefsson, B. (1987). Separation of amino acid Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new enantiomers and chiral amines using precolumn derivatization method for reconstructing phylogenetic trees. Mol Biol Evol4, with ( + )-1 -(9-fluorenyl)ethyl chloroformate and reversed- 406-425. phase liquid chromatography. Anal Chem 59, 1191-1 195. Schleifer, K. H. & Kandler, 0. (1972). Peptidoglycan type of Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric bacterial cell walls and their taxonomic implications. Bacteriol deoxyribonucleic acid-deoxyribonucleic acid hybridization in Rev 36,407-477. microdilution wells as an alternative to membrane filter Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. hybridization in which radioisotopes are used to determine (1996). Agromyces mediolanus sp. nov., nom. rev., comb. nov., a genetic relatedness among bacterial strains. Int J Syst Bacteriol species for ‘ Corynebacterium mediolanum’ Mamoli 1939 and 39,224-229. for some aniline-assimilating bacteria which contain 2,4- diaminobutyric acid in the cell wall peptidoglycan. Int J Syst Fiedler, F. & Kandler 0. (1973). Die Aminosauresequenz von 2,4- Bacteriol46, 88-93. diaminobuttersaure enthaltenden mureinen bei verschiedenen coryneformen bakterien und Agromyces ramosus. Arch Mikro- Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. biol89, 51-66. (1997). Cryobacterium psychrophilum gen. nov., sp. nov., nom. rev., comb. nov., an obligately psychrophilic actinomycete to Fiedler, F., Schleifer, K. H., Cziharz, B., Interschick, E. & Kandler, accommodate ‘ Curtobacterium psychrophilum ’ Inoue and 0. (1 970). Murein types in Arthrobacter, Brevibacteria, Coryne- Komagata 1976. Int J Syst Bacteriol47,474-478. bacteria and Microbacteria. Pub1 Fac Sci Univ J E Purkyne Takahashi, Y., Iwai, Y., Tomoda, H., Nimura, N., Kinoshita, T. & Brno 47, 11 1-122. Omura, S. (1989). Optical resolution of 2,6-diaminopimelic acid Gledhill, W. E. & Casida, L. E., Jr (1969). Predominant catalase- stereoisomers by high performance liquid chromatography for negative soil bacteria. Appl Microbioll7, 208-213. the chemotaxonomy of actinomycete strains. J Gen Appl Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. Microbiol35, 27-32. (1996). Agrococcusjenensis gen. nov., sp. nov., a new genus of Takeuchi, M., Weiss, N., Schumann, P. & Yokota, A. (1996). actinomycetes with diaminobutyric acid in the cell wall. Int J Leucobacter komagatae gen. nov., sp. nov., a new aerobic Syst Bacteriol46, 234-239. Gram-positive, nonsporulating rod with 2,4-diaminobutyric acid in the cell wall. Int J Syst BacterioE46, 967-971. Hayashi, T. & Sasagawa, T. (1993). A method for identifying the carboxy terminal amino acid of protein. Anal Biochem 209, Tamaoka, J. & Komagata, K. (1984). Determination of DNA base 163-168. composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125-1 28. Hensel, R. (1984). Three new murein types in coryneform bacteria isolated from activated sludge. Syst Appl Microbiol5, Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL 11-19. w : Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap Inoue, K. & Komagata, K. (1976). Taxonomic study on obligately penalties and weight matrix choice. Nucleic Acids Res 22, psychrophilic bacteria isolated from Antarctica. J Gen Micro- 4673-4680. biol22, 165-1 76. Tisdall, P. A. & Anhalt, 1. P. (1979). Rapid differentiation of Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in Streptomyces from Nocardia by liquid chromatography. J Clin bacterial systematics. Methods MicrobioZ 19, 161-208. Microbiol 10, 503-505. Leifson, E. (1962). The bacterial flora of distilled and stored Yamada, K. & Komagata, K. (1972a). Taxonomic studies on water. 111. New species of the genera Corynebacterium, Flavo- coryneform bacteria IV. Morphological, cultural, biochemical bacterium, Spirillum and Pseudomonas. Int Bull Bacteriol and physiological characteristics. J Gen Appl Microbiol 18, Nomencl Taxon 12, 161-170. 399-416. Mamoli, L. (1939). Uber biochemische Dehydrierungen in der Yamada, K. & Komagata, K. (197213). Taxonomic studies on Cortingruppe. Ber Dtsch Chem Ges 72, 1863-1865. coryneform bacteria V. Classification of Coryneform bacteria. J Gen Appl Microbioll8, 417-431. Puchala, R., Pior, H., Kulasek, G. W. & Shelford, J. A. (1992). Determination of diaminopimelic acid in biological materials Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N., Voyevoda, using high-performance liquid chromatography. J Chromatogr H. V., Dobrovolskaya, T. G., Lysak, L. V. & Kalakoutskii, L. V. 623, 63-67. (1992). Emended description of the genus Agromyces and description of Agromyces cerinus subsp. cerinus sp. nov., subsp. Rainey, F., Weiss, N., Prauser, H. & Stackebrandt, E. (1994). nov., Agromyces cerinus subsp. nitratus sp. nov., subsp. nov., Further evidence for the phylogenetic coherence of actino- Agromyces fucosus subsp. fucosus sp. nov., subsp. nov., Agro- mycetes with group B-peptidoglycan and evidence for the mycesfucosus subsp. hippuratus sp. nov., subsp. nov. Int J Syst phylogenetic intermixing of the genera Microbacterium and Bacteriol42, 635-641. Aureobacterium as determined by 16s rDNA analysis. FEMS Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N. & Kalakoutskii, Microbiol Lett 118, 135-140. L. V. (1993). Rathayibacter gen. nov., including the species Riley, 1. T. & Ophel, K. M. (1992). Clavibacter toxicus sp. nov., the Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. bacterium responsible for annual ryegrass toxicity in Australia. nov., Rathayibacter iranicus comb. nov., and six strains from Int J Syst Bacteriol42, 64-68. annual grasses. Int J Syst Bacteriol43, 143-149.

41 0 In ternationa I lo urnaI of Systematic Bacteriology 48