Comparative sequence analysis of Mycobacterium leprae and the new leprosy-causing Mycobacterium lepromatosis

Abstract

Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged approximately 10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae.

FIG. 1.

Maximum likelihood phylogenies of selected mycobacteria based on (A) concatenated alignment of the rpoB, ligA, groEL, gnd1, and bfrA genes, (B) alignment of the rpoB gene, and (C) alignment of the 16S rRNA gene. Bootstrap values are indicated at the nodes. At some nodes there are two values, a value obtained by the neighbor-joining method (left value) and a value obtained by the maximum likelihood method (right value). The bars indicate the numbers of nucleotide substitutions per site. The trees were rooted to separate the slow-growing mycobacterial clade (A and B) and the M. avium branch (C).

FIG. 2.

Numbers of nucleotide substitutions per site for five pairs of Mycobacterium species. (Left panel) dS values. (Right panel) dN values. The genes analyzed were rpoB, ligA, groEL, gnd1, and bfrA (bars from left to right). Abbreviations: map, M. avium subsp. paratuberculosis; mav, M. avium 104; mbo, M. bovis AF2122/97; mle, M. leprae; mlp, M. lepromatosis; mmi, M. marinum M; mtc, M. tuberculosis CDC1551; mul, M. ulcerans. The dN/dS ratios for the rpoB, ligA, groEL, gnd1, and bfrA genes for the M. lepromatosis-M. leprae pair were 0.022, 0.155, 0.031, 0.075, and 0.089, respectively; the average dN/dS ratio was 0.074.