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Volume 155, Issue 6

Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase genes in a recombination-impaired Free

Abstract

The human pathogen produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (, 51 kb; , 7.2 kb; , 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome / shuttle vectors, one based on the pMUM001 origin of replication bearing , and the other based on the mycobacteriophage L5 integrase, bearing and . The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to , a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of . To improve the stability of the locus in , was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the Δ mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the genes in both wild-type and the mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.

  • Received:
  • Accepted:
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  • Published Online:
Keyword(s): BAC, bacterial artificial chromosome , LM, load module , Mls, mycolactone polyketide synthase , MPM, mycolactone-producing mycobacteria , PKS, polyketide synthase , qRT-PCR, quantitative RT-PCR and WT, wild-type
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2009-06-01
2024-04-24
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Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum
Microbiology 155, 1923 (2009); https://doi.org/10.1099/mic.0.027029-0
/content/journal/micro/10.1099/mic.0.027029-0
/content/journal/micro/10.1099/mic.0.027029-0
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