Comparative genomic and phylogenetic approaches to characterize the role of genetic recombination in mycobacterial evolution

Abstract

The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.

Figure 1. Trees of genus Mycobacterium based on 16S rRNA gene sequences (a) and concatenated core regions (b).

Trees were inferred using FastTree (a) and concatenated core regions of the genome excluding all recombination events, inferred using RaxML (b). Internal node labels in (a) indicate SH-based “local support” values, and the division between “slow growing” and fast growing” mycobacteria is indicated with a dashed line. All bootstrap support values in (b) were either 99% or 100%, and are consequently not shown.

Figure 2. Percentage of protein sets from each mycobacterial strain assigned to Groups I-IV.

Strains are grouped according to the four main divisions identified in the text.

Figure 3. Recovery of cohesive mycobacterial clans.

The number of trees for proteins in Groups II, III and IV from each mycobacterial genome that recovered genus Mycobacterium as a clan (dark grey) or as multiple groups interspersed with proteins from other genera (light grey) is shown.

Figure 4. Closest-neighbor analysis of trees of proteins from four mycobacterial genomes.

Each row corresponds to a different genome. The left column (a, c, e, g) summarizes the taxonomic composition of the most taxonomically limited neighboring group for all trees containing proteins from a particular organism, while the right-hand column shows the same information for trees containing proteins whose distribution among the mycobacteria is limited to the target species (or complex, in the case of M. tuberculosis). Red bars indicate taxonomic groups that are completely contained within suborder Corynebacterinae, blue bars show groups that have no overlap with Corynebacterinae, and grey bars indicate groups that contain both Corynebacterinae and non-Corynebacterinae.

Figure 5. Patristic distances for genes from selected pairs of genomes.

Distribution of patristic distances (in substitutions per site) between protein sequences from ‘target’ mycobacteria (a, M. abscessus; b, M. smegmatis; c, M. vanbaalenii), non-mycobacterial genera with strong affinities to the target mycobacterium, and ‘calibrating’, closely related mycobacteria. Blue bars show the distribution of distances from sequences found in the target species and no other mycobacteria to the non-mycobacterial group; purple bars show a similar distribution for sequences present in other mycobacterial as well; and black bars show the distribution of distances between the target and calibrating mycobacterial genomes.

Figure 6. Putative function and genomic context of putative laterally transferred genes.

(a, c) distribution of putative transfers according to COG category for pairings of Segniliparus with M. abscessus (a), and Streptomyces with M. smegmatis (c). Alternating shades of blue indicate metabolic categories, green indicates cellular processes, red indicates information storage and processing, and hypothetical and unclassified proteins are shown in gray. (b,d) examples of sets of putatively transferred genes showing conserved linkage. Colors indicate different COGs with distinct IDs. Locus names and corresponding gene names are shown, and the numeric portion of the first and last locus IDs is matched to the corresponding locus in the mycobacterial chromosomal segment.