. AND HANS-JURGEN BUSSE'"Polyamine patterns of 75 strains of actinobacteria belonging to the genera Agrococcus, Agromyces, Aureobacterium, Brevibacterium, Cluvibacter, Corynebacterium, Curtobacterium, Microbacterium, Rathayibacter, and Tsukamurellu 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. In recent years analysis of polyamine patterns has become well-established in the systematics of gram-negative bacteria, and its suitability for classification has been demonstrated for the class Proteobacteria (2-4, 6-8, 20, 22, 25, 26, 28, 30, 49, 50) and the Flavobacteriurn-Cytophaga-Sphingobacterium branch (23, 24). Attempts to introduce polyamine patterns as chemotaxonomic markers for gram-positive bacteria, such as bacilli and lactic acid bacteria (18), thermophilic bacilli (19), actinomycetes (21), and aerobic (18) and anaerobic cocci (27), did not demonstrate the suitability of this approach for the classification of these groups. However, studies on the polyamine contents of the heterogeneous group that includes the actinobacteria have not been performed previously.In the past, actinobacteria, which are also designated the coryneform bacteria, have often been misclassified, which has led to reclassifications or descriptions of new taxa. In the search for a reliable classification and identification system, the actinobacteria have been the subject of numerous investigations, including numerical taxonomic studies (31,42) and analyses of cell wall sugars (47), peptidoglycan types and diamino acids (41), fatty acids (5,11,29,35), mycolic acids (1,34), polar lipids (12, 35), quinones (lo), and 16s rRNA sequences (9,32, 33, 37-40, 43, 48). The combination of these different approaches has led to an improved taxonomy of actinobacteria, although there are many actinobacterial genera which exhibit similar chemotaxonomic features. This study was initiated to evaluate the suitability of polyamine patterns for differentiating actinobacterial taxa. The results of our analysis of the polyamine patterns obtained for different actinobacterial genera are presented below; this analysis included the genera Agrococcu...

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A Gram-positive, aerobic, spherical actinobacterium, designated strain 4-0(T), was isolated from a medieval wall painting and characterized to determine its taxonomic position. The peptidoglycan of strain 4-0(T) was of type A4alpha, with lysine as the diagnostic cell wall diamino acid and an interpeptide bridge of Lys-Gly-Glu. Its quinone system contained predominantly MK-9(H2) (64%) and its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, four unknown glycolipids, two unknown phospholipids and an unknown lipid. The fatty acid profile of strain 4-0(T) was represented by significant amounts of ai-C15:0 and moderate amounts of ai-C17:0, i-C16:0 and i-C15:0 fatty acids. Spermidine was predominant in the polyamine pattern. The G+C content of the genomic DNA was 68 mol%. Comparative 16S rDNA sequence studies revealed the highest similarity values (95.4-96.1%) between strain 4-0(T) and species of the genus Micrococcus and certain species of the genus Arthrobacter, including Arthrobacter pascens DSM 20545(T), Arthrobacter ramosus DSM 20546(T), Arthrobacterprotophormiae DSM 20168(T), Arthrobacternicotianae DSM 20123(T) and Arthrobacter globiformis DSM 20124(T). Phylogenetic analyses demonstrated that strain 4-0(T) branches deeply on the Micrococcus lineage. Because it is almost equidistant phylogenetically from the genera Micrococcus and Arthrobacter and possesses significant differences in chemotaxonomic characteristics from members of these genera, it is suggested that strain 4-0(T) be classified as a novel species in a new genus; the name Citricoccus muralis gen. nov., sp. nov. is proposed. The type strain is strain 4-0(T) (= DSM 11442(T) CCM 4981(T)).

Two bacterial strains, designated 1A-C(T) and 3A-1, were studied and, using these results and previously published data, taxonomically classified. Cells of the strains exhibited a rod-coccus cycle. The peptidoglycan determined for 1A-C(T) was of type A4alpha with lysine as the diagnostic cell-wall diamino acid and an interpeptide bridge of L-Lys <-- L-Glu. The menaquinone systems of the two strains contained MK-8(H4) (82-94%) and MK-7(H4) (3-11%). The polar lipid profiles consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, two unidentified phospholipids and an unidentified glycolipid. The fatty acid profiles contained predominantly ai-C15:0 and significant amounts of i-C14:0 and i-C15:1 fatty acids. Genomic fingerprints clearly distinguished strains 1A-C(T) and 3A-1 from each other. DNA-DNA relatedness between the two strains (92%) demonstrated that they are members of a single species. Analyses of the 16S rDNA sequences of strains IA-C(T) and 3A-1, which were almost identical (99.6% sequence similarity), and comparison with corresponding sequences demonstrated that they represent a novel lineage within the suborder Micrococcineae, most closely related to species of the genera Beutenbergia, Bogoriella and Cellulomonas (94.7-95.7% sequence similarity). The results demonstrate that the two strains are members of a single new genus and a single novel species. Thus, the name Georgenia muralis gen. nov., sp. nov. is proposed. The type strain is strain 1A-C(T) (= DSM 14418T = CCM 4963T). Another strain of the species is strain 3A-1 (= DSM 14419 = CCM 4964).