Key Transitions in the Evolution of Rapid and Slow Growing Mycobacteria Identified by Comparative Genomics

Abstract

Mycobacteria have been classified into rapid and slow growing phenotypes, but the genetic factors that underlie these growth rate differences are not well understood. We compared the genomes of 157 mycobacterial species, representing all major branches of the mycobacterial phylogenetic tree to identify genes and operons enriched among rapid and slow growing mycobacteria. Overlaying growth phenotype on a phylogenetic tree based on 304 core genes suggested that ancestral mycobacteria had a rapid growth phenotype with a single major evolutionary separation into rapid and slow growing sub-genera. We identified 293 genes enriched among rapid growing sub-genera, including genes encoding for amino acid transport/metabolism (e.g., livFGMH operon) and transcription, as well as novel ABC transporters. Loss of the livFGMH and ABC transporter operons among slow growing species suggests that reduced cellular amino acid transport may be growth limiting. Comparative genomic analysis suggests that horizontal gene transfer, from non-mycobacterial genera, may have contributed to niche adaptation and pathogenicity, especially among slow growing species. Interestingly, the mammalian cell entry (mce) operon was found to be ubiquitous, irrespective of growth phenotype or pathogenicity, although protein sequence homology between rapid and slow growing species was low (<50%). This suggests that the mce operon was present in ancestral rapid growing species, but later adapted by slow growing species for use as a mechanism to establish an intra-cellular lifestyle.

Keywords: Mycobacterium species; comparative genomics; evolution; growth phenotype; phylogenetic analysis.

FIGURE 1

Phylogenetic tree of all well characterised Mycobacterium species. This Maximum likelihood tree of 157 well-characterized Mycobacterium species is based on nucleotide alignment of 304 single copy genes. It shows five distinct sub-genera and indicates that slow growers evolved from more ancestral fast growing species. Bootstrap values are shown on nodes with less than 100% support.

FIGURE 2

Functions related to amino acid transport and transcription are significantly enriched in the rapid growing mycobacteria. This plot depicts the number of gene clusters that are classified in each Cluster of Orthologous Group (COG) category within both fast-growing (blue) and slow-growing (red) Mycobacteria. ^∗P-value <0.05 as calculated via Fisher Exact Test; COGs classes without ^∗ are not significantly different between rapid and slow growing species.

FIGURE 3

Four operons related to amino acid and ion transport are enriched within rapid growing mycobacteria. The left-hand panel depicts the same phylogeny as in Figure 1. The right panel displays a heatmap showing the presence (dark blue) or absence (light blue) of genes in the four operons. The livFGMH operon appears to be missing in M. insubricum, M. brumae, and M. duvalii due to gaps in the assembly.

FIGURE 4

The mce1 operon is highly variable across all five mycobacterial sub-genera. The left-hand panel depicts the phylogeny as in Figure 1. The right panel shows a gradient heatmap based on protein sequence identity for each protein encoded by the mce1 operon using BLASTp.

FIGURE 5

Genomic comparison of selected slow and rapid growing mycobacteria highlights the role of horizontal gene transfer in the evolution of slow growers. The innermost ring depicts the M. tuberculosis H37Rv genome coordinates in kilobases (kbp). The next ring shows the GC Content (black). The remaining rings show BLASTn comparison with H37Rv against five other mycobacterial assemblies (from inner to outer: M. intermedium, M. paratuberculosis, M. smegmatis, M. neoaurum, and M. fortuitum; see section “Materials and Methods”). BLAST matches with nucleotide identity 50–100% are colored, while non-matching regions are white. The outer ring contains annotation of specific regions of difference (ROD) found in M. tuberculosis that likely represent acquisition by horizontal gene transfer.