Multiple-genome comparison reveals new loci for Mycobacterium species identification

Abstract

To identify loci useful for species identification and to enhance our understanding of the population structure and genetic variability of the genus Mycobacterium, we conducted a multiple-genome comparison of a total of 27 sequenced genomes in the suborder of Corynebacterineae (18 from the Mycobacterium genus, 7 from the Corynebacterium genus, 1 each from the Nocardia and Rhodococcus genera). Our study revealed 26 informative loci for species identification in Mycobacterium. The sequences from these loci were used in a phylogenetic analysis to infer the evolutionary relations of the 18 mycobacterial genomes. Among the loci that we identified, rpoBC, dnaK, and hsp65 were amplified from 29 ATCC reference strains and 17 clinical isolates and sequenced. The phylogenetic trees generated from these loci show similar topologies. The newly identified dnaK locus is more discriminatory and more robust than the widely used hsp65 locus. The length-variable rpoBC locus is the first intergenic locus between two protein-encoding genes being used for mycobacterial species identification. A multilocus sequence analysis system including the rpoBC, dnaK, and hsp65 loci is a robust tool for accurate identification of Mycobacterium species.

FIG. 1.

(A) Neighbor-joining tree of 26 CHRs from 18 mycobacterial genomes rooted with N. farcinica IFM 10152. Two M. bovis and four M. tuberculosis isolates are compressed into MTBC. The percentages of replicate trees in a bootstrap test of 2,000 replicates are shown at the branches. The complete deletion option for gaps is used. (B) Expanded subtree of MTBC.

FIG. 2.

Single-gene neighbor-joining phylogenetic trees rooted with N. farcinica IFM 10152. (A) rpoBC; (B) dnaK; (C) hsp65. The percentages of bootstrap values are shown next to the nodes. Examples of SGM misplaced into RGM are marked with an asterisk at the end of their names.

FIG. 2.

Single-gene neighbor-joining phylogenetic trees rooted with N. farcinica IFM 10152. (A) rpoBC; (B) dnaK; (C) hsp65. The percentages of bootstrap values are shown next to the nodes. Examples of SGM misplaced into RGM are marked with an asterisk at the end of their names.

FIG. 2.

Single-gene neighbor-joining phylogenetic trees rooted with N. farcinica IFM 10152. (A) rpoBC; (B) dnaK; (C) hsp65. The percentages of bootstrap values are shown next to the nodes. Examples of SGM misplaced into RGM are marked with an asterisk at the end of their names.

FIG. 3.

Neighbor-joining phylogenetic tree of concatenated rpoBC, dnaK, and hsp65 loci. The tree is rooted with N. farcinica IFM 10152. The slowly growing species M. triviale has a unique phylogenetic position and is marked with an asterisk. ^T, type strain.