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Polyamine Distribution in Actinomycetes with Group B

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Polyamine Distribution in Actinomycetes with Group B INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,Apr. 1997, p. 270-277 Vol. 47, No. 2 0020-7713/97/$04.00+0 Copyright 0 1997, International Union of Microbiological Societies Polyamine Distribution in Actinomycetes with Group B Peptidoglycan and Species of the Genera Brevibacterium, Corynebacterium, and Tsukamurella PETRA ALTENBURGER,' PETER €L&MPFER,2 VLADIMIR N. AKIMOV,3 WERNER LUBITZ,' . AND HANS-JURGEN BUSSE'" Institut fur Mikrobiologie uled Genetik, Universitat Wien, A-1030 Vienna, Austria I; Institut fir Angewandte Mikrobiologie, Justus-Liebig-Universitat Giessen, 0-35390 Giessen, Germany2;and All-Russian Collection of Microorganisms, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia3 Polyamine patterns of 75 strains of actinobacteria belonging to the genera Agrococcus, Agromyces, Aureobac- terium, Brevibacterium, Cluvibacter, Corynebacterium, Curtobacterium, Microbacterium, Rathayibacter, and Tsuka- murellu were analyzed in order to investigate the suitability of this approach for differentiation within this group. The results revealed that the overall polyamine contents differ significantly among genera and that various patterns are present in actinobacteria. One characteristic pattern found in the genera Clavibacter, Rathayibacter, and Curtobacterium included a high polyamine concentration, and the polyamines were mainly spermidine and spermine. This feature distinguished the 2,4-diaminobutyric acid-containing genera Rathayibacter, Clavibacter, and Agromyces, which contained low concentrations of polyamines. Strains of the genus Brevibacterium were characterized by the presence of high concentrations of cadavarine and usually high concentrations of putrescine. Members of the genus Corynebacterium had relatively low polyamine contents, and usually spermidine was the major polyamine. A similar polyamine pattern was detected in the species of the genus Tsukamurella. No homogeneous polyamine patterns were detected in representatives of the genera Microbacterium and Aureobacterium, which are phylogenetically intermixed (M. Takeuchi and A. Yokota, FEMS Microbiol. Lett. 124:ll-16, 1994). The results of polyamine analyses are in good agreement with the genetic heterogeneity within the actinobacteria and demonstrate that polyamine patterns are suitable for use in classification of actinobacterial taxa. In recent years analysis of polyamine patterns has become This study was initiated to evaluate the suitability of poly- well-established in the systematics of gram-negative bacteria, amine patterns for differentiating actinobacterial taxa. The re- and its suitability for classification has been demonstrated for sults of our analysis of the polyamine patterns obtained for the class Proteobacteria (2-4, 6-8, 20, 22, 25, 26, 28, 30, 49, 50) different actinobacterial genera are presented below; this anal- and the Flavobacteriurn-Cytophaga-Sphingobacteriumbranch ysis included the genera Agrococcus, Agrornyces, Aureobacte- (23, 24). Attempts to introduce polyamine patterns as chemo- riurn, Brevibacteriurn, Clavibacter, Curtobacterium, Corynebac- taxonomic markers for gram-positive bacteria, such as bacilli terium, Microbacterium, Rathayibacter, and Tsukarnurella. and lactic acid bacteria (18), thermophilic bacilli (19), actino- mycetes (21), and aerobic (18) and anaerobic cocci (27), did MATERIALS AND METHODS not demonstrate the suitability of this approach for the classi- fication of these groups. However, studies on the polyamine Strains used and culture conditions. The designations of the strains analyzed are listed in Tables 1 through 3 The cells were cultivated at 28°C on PYE contents of the heterogeneous group that includes the acti- medium (0.3% peptone from casein, 0.3% yeast extract) and were harvested at nobacteria have not been performed previously. a density corresponding to approximately 70% of the maximum optical density In the past, actinobacteria, which are also designated the determined for each strain. Since Microbacterium luevaniformuns DSM 201407' coryneform bacteria, have often been misclassified, which has (T = type strain), Tsukamurellu inchoensis DSM 44067T, and Tsukamurella wrutisluviensis DSM 44107T did not grow in homogeneous suspensions, the point led to reclassifications or descriptions of new taxa. In the of harvest was estimated. To grow Cluvibacter michigunensis subsp. michigunensis search for a reliable classification and identification system, the ICMP 1808 in mineral medium, 0.3 g of (NH4),S04, 1.04 g of K,HPO,. 0.75 g of actinobacteria have been the subject of numerous investiga- KH,PO,, 0.025 g of CaCI,, and 0.2 g of MgSO, were dissolved in 1 liter of tions, including numerical taxonomic studies (31,42) and anal- glass-distilled water, and the medium was supplemented with 3 ml of a trace metal mixture (36) and 10 mM NH, succinate. The mineral medium used to grow yses of cell wall sugars (47), peptidoglycan types and diamino Corynebacterium glutumicum contained 8 g of K2HP0, per liter, 3.2 g of acids (41), fatty acids (5,11,29,35), mycolic acids (1,34), polar NaH,P04 per liter, 0.2 g of Na, citrate 2H,O per lilter, 2 mg of FeSO, per liter, lipids (12, 35), quinones (lo), and 16s rRNA sequences (9,32, 1.1 g of (NH,),SO, per liter, 0.1 g of MgSO, * 7H,O per liter, and 10 mM 33, 37-40, 43, 48). The combination of these different ap- glucose. Polyamine analysis. Polyamines were extracted as described previously (6). proaches has led to an improved taxonomy of actinobacteria, Approximately 40 mg of lyophilized cells was hydrolyzed in 1 ml of 0.2 N although there are many actinobacterial genera which exhibit perchloric acid for 30 min at 100°C with occasional shaking. Each mixture similar chemotaxonomic features. contained the internal standard 1,8-diaminooctane (0.5 p.moV4O mg of cells). After extraction the samples were centrifuged, and 0.2 ml of the supernatant was transferred to a tube containing 0.3 ml of a Na,CO, solution (100 mg/ml). Then 0.8 ml of a dansyl chloride solution (7.5 pg/ml in acetone) was added. The tube was tightly closed, and dansylation was performed for 20 min at 60°C. Subse- * Corresponding author. Mailing address: Institut fur Mikrobiologie quently, 0.1 ml of a proline solution (SO mg/ml) was added to bind excessive und Genetik, Universitat Wien, Dr. Bohr-Gasse 9, A-1030 Vienna, dansyl chloride during incubation for 10 min at 60°C. After cooling to 5"C, the Austria. Phone: 43-1-79515-4121. Fax: 43-1-79515-4114. E-mail: elliot polyamines were extracted with 0.1 ml of toluene with shaking. The upper phase @gem.univie.ac. a t . was used for high-performance liquid chromatography (HPLC) analysis. The 270 VOL. 47, 1997 POLYAMINE DISTRIBUTION IN ACTINOBACTERIA 271 TABLE 1. Polyamine patterns of peptidoglycan type B and 2,4-diaminobutyric acid-containing actinobacteriaa ~ ~ ~ ~ ~~~~ ~ Polyamine content (Fmol/g [dry ~t])~ Organism DAP PUT CAD NSPD SPD HSPD SPM TYR Total Agrococcus jenensis DSM 9580T 0.03 (3.19) 0.04 (4.25) Tr 0.87 (92.6) Tr 0.94 Agromyces cerinus subsp. ceri- 0.10 (38.5) 0.11 (42.3) 0.02 (7.7) 0.02 (7.7) 0.01 (3.8) 0.26 nus VKM Ac-1340T (= DSM 8595T) Agromyces cerinus subsp. nitra- 0.14 (63.6) 0.01 (4.5) 0.03 (13.6) 0.04 (18.2) 0.22 tus VKM Ac-1351T (= DSM 8596T) Agromyces fucosus subsp. fuco- 0.05 (23.8) 0.12 (57.1) 0.01 (4.8) 0.02 (9.5) 0.01 (4.8) 0.21 sus VKM A~-1345~(= DSM 8597T) Agromyces fucosus subsp. hippu- Tr 0.14 (58.3) Tr 0.07 (29.2) Tr 0.03 (12.5) 0.24 ratus VKM A~-1352~(= DSM 8598T) Agromyces mediolanus DSM 0.07 (25.0) 0.21 (75.0) Tr Tr Tr Tr 0.28 20152T (= ATCC 14004T = NCIB 7206T) (formerly “Coiynebacterium mediola- num”) Agromyces sp. strain DL-89 0.33 (51.6) 0.02 (3.1) 0.07 (10.9) 0.12 (18.6) 0.08 (12.5) 0.62 Aureobacterium barkeri DSM 0.15 (4.8) 2.92 (95.2) 3.07 20145T (= ATCC 15954T = NCIB 9658T) Aureobacterium liquefaciens 0.19 (33.3) 0.02 (3.5) 0.30 (52.6) 0.06 (10.5) 0.57 DSM 20638T (= NCIB 11509T) Aureobacterium testaceum DSM 0.01 (20.0) 0.01 (29.0) 0.02 (40.0) 0.01 (20.0) 0.05 20166T (= ATCC 15829T) “Brevibacterium helvolum” WS 1.29 (23.4) 4.08 (73.8) 0.02 (0.4) 0.13 (2.4) 5.52 1753 (= ATCC 13715 = DSM 20419) Clavibacter michiganensis subsp. 0.04 (1.6) 0.05 (2.1) 0.50 (20.6) 0.87 (35.8) 0.94 (38.7) 0.03 (1.2) 2.43 michiganensis ICMP 2550T (= NCPPB 2979T) Clavibacter michiganensis subsp. 0.09 (4.5) 0.05 (2.5) 0.57 (28.4) 0.60 (29.8) Tr 0.70 (34.8) 2.01 michiganensis ICMP 2539 (= NCPPB 2034) Clavibacter michiganensis subsp. 0.21 (5.3) 0.19 (4.8) 1.65 (41.8) Tr 1.90 (48.1) 3.95 michiganensis ICMP 1808 Clavibacter michiganensis subsp. 0.04 (0.3) 14.60 (99.6) 0.02 (0.1) 14.66 michiganensis ICMP 1808‘ Clavibacter michiganensis subsp. 0.17 (4.7) 0.37 (10.2) 1.55 (42.8) 1.36 (37.6) 0.17 (4.7) 3.62 michiganensis ICMP 5026 Clavibacter michiganensis subsp. 0.11 (10.0) 0.19 (17.3) 0.55 (50.0) 0.25 (22.7) 1.10 michiganensis ATCC 4450 Clavibacter michiganensis subsp. 0.08 (1.8) 0.14 (3.2) 0.07 (1.6) 0.04 (0.9) 1.85 (42.3) 2.19 (50.1) 4.37 insidiosus ICMP 2621T (= NCPPB 1109T) Clavibacter michiganensis subsp. 0.06 (2.2) 0.24 (9.0) 0.20 (7.5) 1.67 (62.3) 0.44 (16.4) 0.07 (2.6) 2.68 insidiosus ICMP 4 192 Clavibacter michiganensis subsp. 0.03 (0.5) 0.09 (1.6) 0.22 (3.8) Tr 2.10 (36.2) 3.36 (57.9) Tr 5.80 insidiosus ICMP 2615 (= NCPPB 1020) Ciavibacter michiganensis subsp .
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