In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56
Graduate School of Biomedical Sciences; Department of Molecular Genetics and Microbiology
Medical Subject Headings
Borrelia burgdorferi; *CpG Islands; *DNA Methylation; DNA, Bacterial; Electroporation; Genetic Complementation Test; Mutagenesis, Insertional; Mutation; Plasmids; Site-Specific DNA Methyltransferase (Cytosine-Specific); *Transformation, Bacterial
Life Sciences | Medicine and Health Sciences
Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies-so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan(r)) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan(r) mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.
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Citation: J Bacteriol. 2008 Dec;190(24):7885-91. Epub 2008 Oct 10. Link to article on publisher's site
Journal of bacteriology
Chen, Qiang; Fischer, Joshua Richard; Benoit, Vivian M.; Dufour, Nicholas P.; Youderian, Philip; and Leong, John M., "In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56" (2008). GSBS Student Publications. 1402.