1887

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.

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2009-06-01
2020-01-25
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References

  1. Betancor L., Fernández M. J., Weissman K. J., Leadlay P. F.. 2008; Improved catalytic activity of a purified multienzyme from a modular polyketide synthase after coexpression with Streptomyces chaperonins in Escherichia coli . ChemBioChem9:2962–2966
    [Google Scholar]
  2. Brosch R., Gordon S. V., Billault A., Garnier T., Eiglmeier K., Soravito C., Barrell B. G., Cole S. T.. 1998; Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect Immun66:2221–2229
    [Google Scholar]
  3. Dussault A. A., Pouliot M.. 2006; Rapid and simple comparison of messenger RNA levels using real-time PCR. Biol Proced Online8:1–10
    [Google Scholar]
  4. Fu J., Wenzel S. C., Perlova O., Wang J., Gross F., Tang Z., Yin Y., Stewart A. F., Müller R., Zhang Y.. 2008; Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res36:e113
    [Google Scholar]
  5. Gunesekere I. C., Kahler C. M., Ryan C. S., Snyder L. A., Saunders N. J., Rood J. I., Davies J.. 2006; Ecf, an alternative sigma factor from Neisseria gonorrhoeae , controls expression of msrAB , which encodes methionine sulfoxide reductase. J Bacteriol188:3463–3469
    [Google Scholar]
  6. Hatfull G. F., Sarkis G. J.. 1993; DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol Microbiol7:395–405
    [Google Scholar]
  7. Hong H., Spencer J. B., Porter J. L., Leadlay P. F., Stinear T.. 2005; A novel mycolactone from a clinical isolate of Mycobacterium ulcerans provides evidence for additional toxin heterogeneity as a result of specific changes in the modular polyketide synthase. ChemBioChem6:643–648
    [Google Scholar]
  8. Hu Y., Coates A. R.. 1999; Transcription of two sigma 70 homologue genes, sigA and sigB , in stationary-phase Mycobacterium tuberculosis . J Bacteriol181:469–476
    [Google Scholar]
  9. Johnson P. D., Stinear T., Small P. L., Pluschke G., Merritt R. W., Portaels F., Huygen K., Hayman J. A., Asiedu K.. 2005; Buruli ulcer ( M. ulcerans infection): new insights, new hope for disease control. PLoS Med2:e108
    [Google Scholar]
  10. Lea-Smith D. J., Pyke J. S., Tull D., McConville M. J., Coppel R. L., Crellin P. K.. 2007; The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan. J Biol Chem282:11000–11008
    [Google Scholar]
  11. Martinez A., Kolvek S. J., Yip C. L., Hopke J., Brown K. A., MacNeil I. A., Osburne M. S.. 2004; Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl Environ Microbiol70:2452–2463
    [Google Scholar]
  12. Mve-Obiang A., Lee R. E., Umstot E. S., Trott K. A., Grammer T. C., Parker J. M., Ranger B. S., Grainger R., Mahrous E. A., Small P. L.. 2005; A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin. Infect Immun73:3307–3312
    [Google Scholar]
  13. Paget E., Davies J.. 1996; Apramycin resistance as a selective marker for gene transfer in mycobacteria. J Bacteriol178:6357–6360
    [Google Scholar]
  14. Papavinasasundaram K. G., Colston M. J., Davis E. O.. 1998; Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function. Mol Microbiol30:525–534
    [Google Scholar]
  15. Parish T., Stoker N. G.. 2000; glnE is an essential gene in Mycobacterium tuberculosis . J Bacteriol182:5715–5720
    [Google Scholar]
  16. Pidot S. J., Hong H., Seemann T., Porter J. L., Yip M. J., Men A., Johnson M., Wilson P., Davies J. K.. other authors 2008; Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones. BMC Genomics9:462
    [Google Scholar]
  17. Ranger B. S., Mahrous E. A., Mosi L., Adusumilli S., Lee R. E., Colorni A., Rhodes M., Small P. L.. 2006; Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F. Infect Immun74:6037–6045
    [Google Scholar]
  18. Staunton J., Weissman K. J.. 2001; Polyketide biosynthesis: a millennium review. Nat Prod Rep18:380–416
    [Google Scholar]
  19. Stinear T. P., Mve-Obiang A., Small P. L., Frigui W., Pryor M. J., Brosch R., Jenkin G. A., Johnson P. D., Davies J. K.. other authors 2004; Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans . Proc Natl Acad Sci U S A101:1345–1349
    [Google Scholar]
  20. Stinear T. P., Hong H., Frigui W., Pryor M. J., Brosch R., Garnier T., Leadlay P. F., Cole S. T.. 2005a; Common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans . J Bacteriol187:1668–1676
    [Google Scholar]
  21. Stinear T. P., Pryor M. J., Porter J. L., Cole S. T.. 2005b; Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans . Microbiology151:683–692
    [Google Scholar]
  22. Stinear T. P., Seemann T., Pidot S., Frigui W., Reysset G., Garnier T., Meurice G., Simon D., Bouchier C.. other authors 2007; Reductive evolution and niche-adaptation inferred from the genome of Mycobacterium ulcerans , the causative agent of Buruli ulcer. Genome Res17:192–200
    [Google Scholar]
  23. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H.. other authors 1991; New use of BCG for recombinant vaccines. Nature351:456–460
    [Google Scholar]
  24. Talaat A. M., Trucksis M.. 2000; Transformation and transposition of the genome of Mycobacterium marinum . Am J Vet Res61:125–128
    [Google Scholar]
  25. Velkov T., Singaretnam L. G., Lawen A.. 2006; An improved purification procedure for cyclosporin synthetase. Protein Expr Purif45:275–287
    [Google Scholar]
  26. Waterfield N. R., Sanchez-Contreras M., Eleftherianos I., Dowling A., Yang G., Wilkinson P., Parkhill J., Thomson N., Reynolds S. E.. other authors 2008; Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts. Proc Natl Acad Sci U S A105:15967–15972
    [Google Scholar]
  27. Weissman K. J., Leadlay P. F.. 2005; Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol3:925–936
    [Google Scholar]
  28. Yip M. J., Porter J. L., Fyfe J. A., Lavender C. J., Portaels F., Rhodes M., Kator H., Colorni A., Jenkin G. A., Stinear T.. 2007; Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor. J Bacteriol189:2021–2029
    [Google Scholar]
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