Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer

Abstract

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

Figure 1.

Circular representation of the Mycobacterium ulcerans 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, replication; 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 IS2404 and IS2606 is then shown (cyan). The following two circles show forward and reverse strand pseudogenes (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.

Figure 2.

Diagram depicting the rearrangements in, and deletions from, the genome of M. ulcerans compared with M. marinum. The figure shows a linear genomic comparison generated with ACT (Carver et al. 2005). (Red lines) Regions of DNA:DNA identity, (blue lines) inverted regions, (orange) strand-displacing rearrangements, (arrow) position of the displaced 71-kb fragment.

Figure 3.

Some important biochemical pathways. The main biochemical activities of M. ulcerans discussed in the text are depicted; these are all present in M. marinum except for mycolactone biosynthesis encoded by pMUM001. Some key genes and pathways that have been inactivated in M. ulcerans are indicated.

Figure 4.

Partial matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectra of lipids from M. ulcerans and M. marinum. These spectra show the presence of diesters of phthiocerol and phthiodiolone, and of phenolic glycolipids in M. marinum, whereas only diesters of phthiodiolone and phenolphthiodiolone occur in M. ulcerans.