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Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans
Timothy P Stinear1,2, Melinda J Pryor3, Jessica L Porter1
1Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, 3800, Australia.
Abstract:
The presence of a 174 kb plasmid called pMUM001 in Mycobacterium ulcerans, the first example of a mycobacterial plasmid encoding a virulence determinant, was recently reported. Over half of pMUM001 is devoted to six genes, three of which encode giant polyketide synthases (PKS) that produce mycolactone, an unusual cytotoxic lipid produced by M. ulcerans. In this present study the remaining 75 non-PKS-associated protein-coding sequences (CDS) are analysed and it is shown that pMUM001 is a low-copy-number element with a functional ori that supports replication in Mycobacterium marinum but not in the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Sequence analyses revealed a highly mosaic plasmid gene structure that is reminiscent of other large plasmids. Insertion sequences (IS) and fragments of IS, some previously unreported, are interspersed among functional gene clusters, such as those genes involved in plasmid replication, the synthesis of mycolactone, and a potential phosphorelay signal transduction system. Among the IS present on pMUM001 were multiple copies of the high-copy-number M. ulcerans elements IS2404 and IS2606. No plasmid transfer systems were identified, suggesting that trans-acting factors are required for mobilization. The results presented here provide important insights into this unusual virulence plasmid from an emerging but neglected human pathogen.
Insights
The Mycobacterium ulcerans virulence plasmid pMUM001 contains genes for mycolactone production and a mosaic structure with insertion sequences. It replicates in M. marinum but not in other mycobacteria, requiring trans-acting factors for transfer.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Mycobacterium ulcerans produces mycolactone, a cytotoxic lipid responsible for virulence.
- The 174 kb plasmid pMUM001 is the first identified mycobacterial plasmid encoding a virulence determinant.
- pMUM001 contains six genes, with three encoding giant polyketide synthases (PKS) for mycolactone synthesis.
Purpose of the Study:
- To analyze the 75 non-PKS-associated protein-coding sequences (CDS) on the pMUM001 plasmid.
- To investigate the replication capabilities and genetic structure of pMUM001.
- To understand the role of pMUM001 in Mycobacterium ulcerans virulence.
Main Methods:
- Sequence analysis of pMUM001 protein-coding sequences (CDS).
- Replication studies in Mycobacterium marinum, Mycobacterium smegmatis, and Mycobacterium fortuitum.
- Identification and characterization of insertion sequences (IS) and gene clusters.
Main Results:
- pMUM001 is a low-copy-number plasmid with a functional origin of replication (ori) supporting replication in M. marinum.
- Replication was not observed in fast-growing mycobacteria (M. smegmatis, M. fortuitum).
- The plasmid exhibits a mosaic gene structure with interspersed insertion sequences (IS), including IS2404 and IS2606, and lacks identified plasmid transfer systems.
Conclusions:
- pMUM001 possesses a complex, mosaic structure indicative of large plasmid evolution.
- Replication and potential mobilization of pMUM001 are likely regulated by host factors.
- These findings offer insights into the virulence plasmid of Mycobacterium ulcerans, an emerging neglected pathogen.
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