Masters Theses

Date of Award

8-2008

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Pamela Small

Committee Members

Todd Reynolds, Chunlei Su

Abstract

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a necrotizing skin disease endemic to West Africa and Australia. The cytopathicity, cell cycle arrest and immunosuppression characteristic of BU are attributed to the production of a plasmid-encoded, macrolide toxin, mycolactone. The core of mycolactone is a product of two large polyketide synthases (PKS) and is conserved among all mycolactone congeners. Heterogeneity of the toxin is a result of differences in the polyketide side chain, the product of a third PKS. The mycolactone plasmid (MP) was initially thought to be restricted to M. ulcerans. However, other mycolactone producing mycobacteria (MPMs) have now been identified in association with diseased frogs and fish. Although plasmids are common in environmental mycobacteria, nothing is known about the ability of these plasmids to participate in horizontal transfer. This work investigates the expression of mycolactone in heterologous hosts, M. fortuitum and M. marinum, as well as the ability of MPMs to participate in conjugation.

In this work a 152 kb fragment of the 154 kb MP plasmid from M. ulcerans 1615 was cloned into pBeloBAC11 and introduced into E. coli. This plasmid, pMYCO7017, contains the mycobacterial plasmid origin of replication as well as the mycolactone gene cluster. A Kanamycin resistance gene was introduced into pMYCO7017 by transposon mutagenesis. A construct, pMYCO7017::TnKm, containing an insertion outside the mycolactone gene cluster, was isolated for further work. Electroduction was used to transfer pMYCO7017:TnKm from its E. coli host into both M. marinum 1218 and a plasmid minus strain of M. fortuitum.

One M. marinum and four M. fortuitum transformants were verified by PCR analysis. Lipids were extracted from the transformants, and then analyzed by TLC and cytotoxicity assay, but results were inconclusive. However, when lipid extracts were analyzed by mass spectrometry and HPLC, a novel molecule was discovered indicating that one transformant, M. fortuitum 10394.6 (pMYCO7017::TnKm) was able to produce the mycolactone core. This work is the first example of heterologous expression of an M. ulcerans mycolactone PKS genes. Importantly, the gene cluster contained on pMYCO7017::TnKm is expressed. These results further suggest that the requirements for synthesis of the mycolactone core differ from those for the side-chain.

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