Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB)

Abstract

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.

FIG. 1

A set of primers (MF-MR) that can amplify mycobacterial rpoB DNA (342 bp) was selected from the rpoB sequences of M. tuberculosis, M. leprae, and M. smegmatis (GenBank accession no. L27989, Z14314, and U24474, respectively) corresponding to highly conserved regions (HCR5 and HCR6) of B. subtilis and E. coli. (A) Primary sequences of the RNA polymerase β subunits of B. subtilis and E. coli are composed of four conserved domains (C1 to C4) and three variable domains (V1 to V3). (B) The rif region (nucleotides 507 to 533 [E. coli numbering]), associated with rifampin resistance in M. tuberculosis, is flanked by HCR5 (nucleotides 444 to 454) and HCR6 (nucleotides 547 to 577).

FIG. 2

Sequence pair distances of 44 reference species of mycobacteria determined by using the Clustal program with weighted residue weight table (MegAlign package [Windows version 3.12e]; DNASTAR, Madison, Wis.). M. tuberculosis complex consists of M. tuberculosis, M. bovis, M. bovis BCG, and M. africanum.

FIG. 3

Deduced amino acid sequences (R₄₅₄ to H₅₅₄ [E. coli numbering]) of rpoB DNAs from 44 reference strains of mycobacteria and 6 nonmycobacterial species. Nucleotide sequences of B. subtilis, S. aureus, E. coli (GenBank accession no. L24376, X64172, and V0040, respectively, were used for comparisons). Asterisks indicate amino acids that are frequently changed in rifampin-resistant M. tuberculosis.

FIG. 4

Phylogenetic tree based on rpoB gene sequences (GenBank accession no. AF057449 to AF057496) shows the relationships of the 44 reference strains of mycobacteria, including M. tuberculosis complex (M. tuberculosis, M. bovis, M. bovis BCG, and M. africanum). ATCC numbers of M. celatum and M. fortuitum strains are shown in parentheses. This tree was constructed by the neighbor-joining method. Topology was also evaluated by bootstrap analysis (MEGA program, 100 repeats, with R. equi as the outgroup). The numerical values in the tree represent bootstrap results. The distance between two strains is the sum of the branch lengths between them.