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Journal of Bacteriology, March 2005, p. 1668-1676, Vol. 187, No. 5
0021-9193/05/$08.00+0     doi:10.1128/JB.187.5.1668-1676.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Common Evolutionary Origin for the Unstable Virulence Plasmid pMUM Found in Geographically Diverse Strains of Mycobacterium ulcerans

Timothy P. Stinear,1,2* Hui Hong,3 Wafa Frigui,1 Melinda J. Pryor,4,{dagger} Roland Brosch,1 Thierry Garnier,1 Peter F. Leadlay,5 and Stewart T. Cole1

Unité de Génétique Moléculaire Bactérienne,1 Plate-Forme 4-Intégration et Analyse Génomiques, Génopole, Institut Pasteur, Paris, France,4 Department of Microbiology, Monash University, Clayton, Australia,2 Department of Chemistry,3 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom5

Received 8 October 2004/ Accepted 29 November 2004

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

* Corresponding author. Present address: Department of Microbiology, Monash University, Wellington Road, Clayton 3800, Australia. Phone: 61-3-99059709. Fax: 61-3-99054811. E-mail: tim.stinear{at}med.monash.edu.au.

{dagger} Present address: Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia.

Journal of Bacteriology, March 2005, p. 1668-1676, Vol. 187, No. 5
0021-9193/05/$08.00+0     doi:10.1128/JB.187.5.1668-1676.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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