Sequencing of the 16s ribosomal DNAs (rDNA) of two strains of Rhodococcus maris was performed to determine the relationship of this species to other mycolic acid-containing actinomycetes. For this purpose we also determined the 16s rDNA sequences for the type species of the genus Rhodococcus, Rhodococcus rhodochrous, and for Mycobacterium chlorophenolicum (formerly Rhodococcus chlorophenolicus) , Rhodococcus erythropolis, Gordona bronchialis, and Gordona terrae, for which only partial sequence data have been available previously. The sequences of the two strains of R. mans were identical. The results of a distance matrix analysis indicated that R. maris is not a member of the genus Rhodococcus but is located between members of the genus Corynebacterium and members of the Rhodococcus-Nocardia-Mycobacterium-Gordonu-Tsukumureh cluster. The finding that R. maris is phylogenetically isolated is supported by the presence of N-acetyl residues in the glycan moiety of the peptidoglycan and the lack of phosphatidylinositol and phosphatidylinositol mannosides, characteristics which distinguish this taxon from related taxa. On the basis of our results and previous findings, we propose that R. maris should be reclassified in a new genus, Dietzia. The type species is Dietzia maris comb. nov.Rhodococcus mans was originally known as "Flavobactenum maris" (9) but later was classified as a species of the genus Rhodococcus (17). The reason for this transfer was the presence of morphological and chemotaxonomic characteristics of the genus Rhodococcus, including gram-positive cells, lack of an aerial mycelium, cell wall chemotype IV, mycolic acids of the Rhodococcus eiythropolis type, MK-8(H2) as the major isoprenolog, fatty acids that included straight-chain saturated and monounsaturated fatty acids and tuberculostearic acid, and a DNA G+C content of 73.2 mol%. Differentiation from other Rhodococcus species was based on the results of physiological reactions. In this study we found that on the basis of additional chemotaxonomic data and phylogenetic evidence, R. mans cannot be considered an authentic member of the genus Rhodococcus. MATERIALS AND METHODSBacterial strains and cultivation. R. mans DSM 43672T (T = type strain) and DSM 46102, R. erythropolis DSM 43066T, Rhodococcus rhodochrous DSM 43241T, Gordona bronchialis DSM 43247T, Gordona terrae DSM 43249T, and Mycobactenum chlorophenolicum (formerly Rhodococcus chlorophenolicus) DSM 43826T were grown on TSB agar (3% [wt/vol] 16s rDNA sequencing. Genomic DNAs were extracted from two strains of R. mans, R. rhodochrous, R. erythropolis, M. chlorophenolicum, Corynebactenum glutamicum, G. bronchialis, and G. terrae and the 16s ribosomal DNAs (rDNAs) were amplified as described previously (20). PCR products were sequenced directly by using a Taq DyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. The sequence reaction products were electrophoresed by using an Applied Biosystems model 373A DNA sequencer. Bootstrap values, which ...
Investigation of fatty acid and mycolic acid properties of certain members of the genera Nocardia and Rhodococcus indicated identical spectra for certain pairs of organisms. Binary 16s rDNA sequence homology for the pairs Rhodococcus rhodochrous and R. roseus, Nocardia calcarea and R. erythropolis, and N. restricta and R. equi ranged between 99.2 and 99.9% for each of these pairs of species. Corresponding DNA-DNA similarity values, obtained by the spectrophotometric method, were significantly higher than 70%. Because certain chemotaxonomic properties and the majority of published phenotypic characters support the high degree of relatedness, we propose to transfer R. roseus to R. rhodochrous. The unambiguous assignment of actinomycete species to the genera Rhodococcus and Nocardia constitutes a problem, because virtually no single characteristic of diagnostic value, such as phage susceptibility (19), cell wall chemistry, fatty acid and phospholipid composition, overall size of mycolic acids, and DNA G + C content, has been found to clearly distinguish between members of the two genera (6). Today, the morphological life cycle and the presence of a cyclic menaquinone of the MK-8(H4) type (1, 9, 15) are the main features that distinguish members of Nocardia from those of Rhodococcus. Recent applications of 16s rDNA sequence analysis, DNA hybridization, and chemotaxonomic analysis to strains of both genera have led to taxonomic rearrangements that excluded certain species from the genus Rhodococcus (2, 8, 13, 21, 23) or showed them to be synonyms (12). In this communication we present further evidence that the present classification of certain additional Nocardia and Rhodococcus species is not supported by their phylogenetic and phenotypic relationships. MATERIALS AND METHODSBacterial strains and cultivation. The following strains were investigated for fatty acid and mycolic acid pattern analysis, DNA reassociation, and 16s rDNA analysis (16s rDNA accession numbers in brackets): Nocurdiu reshicta DSM 43199T (X80613), Rhodococcus rhodochrous DSM 43241T (X79288), R. equi DSM 20307T (X80614), R erythropolis DSM 43066T (X79289), R roseus DSM 43274T (X80624), and R ruber DSM 4333gT (X80625 (17) and from our own entries. Direct pairwise sequence homologies were calculated and are presented as percentage similarity values.DNA isolation and characterization. DNA isolation and DNA-DNA hybridization (4,lO) and determination of renaturation rates (3,ll) were performed by previously described methods.Lipid and mycolic acid analysis. Whole-cell fatty acids and free mycolic acids were analyzed as described previously (8, 14, 18). RESULTS AND DISCUSSION16s rDNA analysis of certain pairs of type strains of Nocardia and Rhodococcus species yielded almost identical results (Table 1). Even at this high level of sequence homology, DNA reassociation values may drop below the level of 70% similarity recommended for identification of separate species (25); therefore, DNA hybridization was carried out for these pairs of strains. In each ca...
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