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Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IMMIB RIV-085T and IMMIB RIV-095 are AJ854055 and AJ854056, respectively.
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).
The genus Rhodococcus is classified in the suborder Corynebacterineae (Stackebrandt et al., 1997) and, together with the genus Nocardia, is included in the family Nocardiaceae. The application of molecular identification methods, particularly 16S rRNA gene sequencing, in parallel with improved phenotypic approaches has not only improved the classification of the rhodococci but also has greatly facilitated the discovery of novel species (Rainey et al., 1995; Goodfellow et al., 1998; McMinn et al., 2000; Gürtler et al., 2004). Phylogenetic trees based on the 16S rRNA gene show that members of the genus Rhodococcus can be assigned to four (Rainey et al., 1995), or even six (Gürtler et al., 2004), subclades. The genus Rhodococcus has undergone considerable expansion in the past few years, and, at the time of writing, 21 species are recognized. In the present study, the taxonomic status of two isolates, IMMIB RIV-085T and IMMIB RIV-095, which were isolated from blood-sucking bugs of the genus Triatoma, was determined using polyphasic taxonomic approaches. On the basis of the genotypic and phenotypic data reported here, the two isolates should be recognized as a novel species of the genus Rhodococcus.
Strains IMMIB RIV-085T and IMMIB RIV-095 were cultured on Columbia agar (Becton Dickinson) supplemented with 5 % sheep blood and brain heart infusion agar (Becton Dickinson) to determine their morphological properties. The strains were biochemically characterized by using tests for the hydrolysis of complex substrates, as described previously (Gordon, 1966, 1967; Gordon & Mihm, 1957), as well as tests to determine carbon-source utilization, according to Yassin et al. (1995). The presence of the isomeric form of diaminopimelic acid was tested by using the methods of Becker et al. (1964), while whole-cell sugars were determined by the method of Lechevalier (1968). Lipids were extracted by using acid methanolysis. Mycolic acids were detected with TLC as described by Minnikin et al. (1980); pyrolysis GC of the mycolate was performed according to Yassin et al. (1993a). Non-hydroxylated fatty acids were purified, identified and quantified by GC as described by Yassin (1988). Menaquinones were extracted, purified and identified according to Collins et al. (1977).
DNA was isolated and purified as described previously (Yassin et al., 2000). DNA–DNA hybridization studies were carried out by using the thermal renaturation method (Yassin et al., 1993b). Genomic DNA extraction, PCR-mediated amplification of 16S rRNA genes and the purification of PCR products were carried out using procedures described previously (Rainey et al., 1996). Purified PCR products were sequenced using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) as described in the manufacturer's protocol. An Applied Biosystems 310 DNA Genetic Analyzer was used for the electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of strains IMMIB RIV-085T and IMMIB RIV-095 as well as those (retrieved from GenBank) of Rhodococcus species with validly published names were added to the ARB database (Ludwig et al., 2004) and aligned using the integrated aligner software in the ARB package. The resulting alignment was corrected manually and evolutionary trees were inferred using maximum parsimony (Kluge & Farris, 1969), neighbour joining (Saitou & Nei, 1987) and maximum likelihood (Felsenstein, 1981). An evolutionary distance matrix was calculated using the corrections of Jukes & Cantor (1969). The tree topology was evaluated according to the results of the neighbour-joining and maximum-likelihood analyses. The robustness of the phyletic lines was evaluated by using bootstrap analyses (Felsenstein, 1985) of neighbour-joining datasets for 1000 resamplings. The phylogenetic analyses were carried out using the ARB package.
Strains IMMIB RIV-085T and IMMIB RIV-095 have morphological properties consistent with their assignment to the genus Rhodococcus. The organisms are aerobic. They produce white to slightly creamy colonies with slightly irregular edges on Columbia agar supplemented with 5 % sheep blood. Initially, the cells are thin and filamentous and stain Gram-positive and slightly acid–alcohol-fast. At a later stage of growth, the filaments fragment into rod- to coccoid-shaped elements characteristic of the genus Rhodococcus. The physiological properties of strains IMMIB RIV-085T and IMMIB RIV-095 are described in detail later, in the species description. Biochemical characteristics that differentiate these strains from some of the Rhodococcus species with validly published names (i.e. those examined in this study) are shown in Table 1.
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). One-dimensional TLC of whole-cell acid methanolysates of strains IMMIB RIV-085T and IMMIB RIV-095 revealed the presence of two lipid spots on the chromatogram. The lower one corresponds to mycolic acids, as identified by its RF value (0·56), and the higher spot corresponds to the non-hydroxylated fatty acids. Pyrolysis GC of the purified mycolic acid methyl esters from strains IMMIB RIV-085T and IMMIB RIV-095 released fatty acid methyl esters of C16 : 0 (63·8 % of total cleavage products) and C18 : 0 (36·1 %) as pyrolysis cleavage products. GC analyses of the non-hydroxylated fatty acid methyl esters revealed the presence of tetradecanoate (2·3 % of total fatty acids), pentadecanoate (0·5 %), cis-hexadecenoate (6·8 %), hexadecanoate (35·5 %), heptadecanoate (1·9 %), octadecenoate (3·6 %), octadecanoate (7·8 %), tuberculostearic acid (10-methyl octadecanoate, 36·5 %), eicosenoate (3·4 %) and eicosanoate (1·2 %) as the major cellular fatty acid methyl esters. Mass-spectral analysis of the respiratory quinones showed that both isolates possess dihydrogenated menaquinones with eight isoprene units, MK-8(H2), as the main isoprenoid quinone. These chemotaxonomic similarities to the Rhodococcus species with validly published names are supported by the high levels of 16S rRNA gene sequence similarity observed between isolates IMMIB RIV-085T and IMMIB RIV-095 and members of the genus Rhodococcus.
To ascertain the phylogenetic position of strains IMMIB RIV-085T and IMMIB RIV-095, their almost-complete 16S rRNA gene sequences [1486 nt; 96·3 % of the Escherichia coli sequence (Brosius et al., 1978)], were determined in this study and subjected to comparative analyses. The 16S rRNA gene sequence comparison showed clearly that strains IMMIB RIV-085T and IMMIB RIV-095 are members of the family Nocardiaceae (Stackebrandt et al., 1997). The high values for the 16S rRNA gene sequence similarity to other previously described members of the genus Rhodococcus (96·4–98·3 %) support the addition of strains IMMIB RIV-085T and IMMIB RIV-095 to this genus. There were significantly lower levels of similarity to other taxa of the Actinomycetales (data not shown). The highest sequence similarity was shown to Rhodococcus wratislaviensis, Rhodococcus rhodnii and Rhodococcus corynebacteroides (98·0, 98·3 and 98·4 % sequence similarity, respectively). The high value of 16S rRNA sequence similarity (100 %) between strain IMMIB RIV-085T and strain IMMIB RIV-095 demonstrates their high level of genealogical relatedness. The unrooted phylogenetic tree shown in Fig. 1 was constructed from neighbour-joining analysis. The results confirmed the association of strains IMMIB RIV-085T and IMMIB RIV-095 with the genus Rhodococcus. It was evident from the tree that isolates IMMIB RIV-085T and IMMIB RIV-095 represent a distinct subline, associated with R. rhodnii, within the genus Rhodococcus. These results suggest that strains IMMIB RIV-085T and IMMIB RIV-095 belong to a genetically distinct Rhodococcus species that is closely related to R. corynebacteroides and R. rhodnii (1·6 and 1·7 % sequence divergence, respectively). In view of the high levels of 16S rRNA sequence similarity between strains IMMIB RIV-085T and IMMIB RIV-095 and R. corynebacteroides and R. rhodnii, chromosomal DNA–DNA hybridization studies were performed to establish whether strains IMMIB RIV-085T and IMMIB RIV-095 represent a distinct species. Strain IMMIB RIV-085T displayed low levels of DNA–DNA reassociation with the R. corynebacteroides type strain, DSM 20151T (32·0 %), and with the R. rhodnii type strain, DSM 43336T (43·0 %); these results are below the cut-off point recommended by Wayne et al. (1987) for the circumscription of bacterial genomic species, and confirm the separation of strains IMMIB RIV-085T and IMMIB RIV-095 from their nearest phylogenetic neighbours. At present, the highest 16S rRNA gene sequence similarity value found between representatives of Rhodococcus species with validly published names is that between Rhodococcus opacus and Rhodococcus percolatus: the type strains of these species have a 16S rRNA gene similarity value of 99·3 % and a DNA–DNA relatedness value of 71 % (Briglia et al., 1996).
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Description of Rhodococcus triatomae sp. nov.
Rhodococcus triatomae (tri.a.to'mae. N.L. gen. n. triatomae of Triatoma, a zoological genus of blood-sucking bug from which the micro-organism was isolated).
Produces white to creamy colonies with slightly irregular edges on Columbia blood agar. In the early growth phase, the cells are thin and filamentous, showing elementary branching. At a later stage of growth, the filaments fragment into rod- to coccoid-shaped elements characteristic of the genus Rhodococcus. The filaments as well as the rod- or coccoid-shaped elements stain Gram-positive and are slightly acid–alcohol-fast. Grows at temperatures in the range 22–42 °C. Has the salient chemotaxonomic characteristics of Rhodococcus. Upon pyrolysis, its mycolic acids release fatty acids C16 : 0 and C18 : 0 as the major cleavage products. The fatty acid profile mainly consists of straight-chain saturated, unsaturated and 10-methyl-branched fatty acids. Hydrolyses adenine and testosterone, but not casein, elastin, aesculin, gelatin, guanine, hypoxanthine, tyrosine, urea or xanthine. Assimilates acetate, citrate and glucose, but not adonitol, adipate, isoamyl alcohol, L-arabinose, 2,3-butanediol, cellobiose, meso-erythritol, galactose, gluconate, m-hydroxybenzoate, p-hydroxybenzoate, myo-inositol, lactate, lactose, maltose, mannitol, melezitose, 1,2-propanediol, raffinose, rhamnose, sucrose, sorbitol, trehalose or xylose, as carbon sources for energy. Utilizes acetamide, but not alanine, gelatin, proline, serine, ornithine or arginine, as simultaneous carbon and nitrogen sources.
The type strain is strain IMMIB RIV-085T (=CCUG 50202T=DSM 44892T). A reference strain is IMMIB RIV-095 (=CCUG 50203=DSM 44893).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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