Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

Abstract

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

Figure 1.

(A) Circular representation of the M. marinum chromosome. The scale is shown in megabases in the outer black circle. Moving inward, the next two circles show forward- and reverse-strand CDS, respectively, with colors representing the functional classification. (Red) Information pathways; (light blue) regulation; (light green) hypothetical protein; (dark green) cell wall and cell processes; (orange) conserved hypothetical protein; (cyan) IS elements; (yellow) intermediate metabolism; (gray) lipid metabolism; (purple) PE/PPE. The location of each copy of a PE or PPE gene is then shown (purple). The following two circles show forward- and reverse-strand genes that are not found in other mycobacteria (colors represent the functional classification), followed by the G+C content and finally the GC skew (G–C)/(G+C) using a 20-kb window. (B) Circular representation of the M. marinum mercury-resistance plasmid pMM23, following the same color scheme as the chromosome map but with a different scale in kilobase pairs as indicated. (C) AT skew analysis generated with Gene-Spaghetti, a method for visualizing DNA composition. The colors reflect AT skew (T – A)/(T + A). (Red) Extreme = 60% T-rich; (blue) extreme = 60% A-rich. The software uses a sliding Gaussian window to estimate local base usage (http://www.vicbioinformatics.com/genespaghetti.shtml). Outlier locus_tags were represented by three types of CDS; (blue) NRP; (gray) PE-PGRS; (red) PPE.

Figure 2.

(A) Module and domain architecture of selected NRPSs. (B) Inferred phylogeny using the Neighbor-Joining method (Saitou and Nei 1987) of selected M. marinum condensation domains based on aligned predicted amino acid sequences. Different colors highlight condensation domains within an operon. Replicate trees in which the associated domains clustered together on <80% of occasions in a bootstrap test (10,000 replicates) are indicated. The scale is the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the data set.

Figure 3.

(A) Orthology frequency plots based on reciprocal best-hit FASTA analysis (refer to Methods) for each pairwise ortholog comparison among six species of mycobacteria. (B) Phylogenomic analysis of six mycobacterial species based on percent amino acid identity among 1072 core, shared orthologs. All nodes had >99.98% bootstrap support. (C) Diagram depicting linear genomic comparisons generated with the BLASTN, and displayed with the Artemis Comparison Tool (Carver et al. 2005) of (listed from top to bottom) M. smegmatis mc²155, M. marinum, M. tuberculosis H37Rv, M. avium subsp. paratuberculosis K10, M. marinum M, and M. ulcerans Agy99. Regions of DNA:DNA similarity are joined by red lines for direct matches, and blue lines for inverted matches.

Figure 4.

Venn diagrams showing orthologous CDS among four mycobacterial species as determined by BLASTCLUST analysis. Numbers in parentheses include paralogous CDS.

Figure 5.

Four examples of LGT in M. tuberculosis H37Rv as predicted by Alien_Hunter (sections highlighted in pink) and whole-genome comparisons with M. marinum and M. avium subsp. paratuberculosis, displayed using the Artemis Comparison Tool. Regions of DNA:DNA similarity are joined by red lines, while inverted regions are shown as blue lines (reversed and complemented here for clarity).