Microbiology & Immunology - Research Publications
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ItemNo Preview AvailableReductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2007-02)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.
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ItemDeciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones(BMC, 2008-10-07)BACKGROUND: Mycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001. RESULTS: We report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone F CONCLUSION: The mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.
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ItemMycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer(PUBLIC LIBRARY SCIENCE, 2009-11)Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.