CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
Fig 6
Evidence for intracellular movement of the IS2-like megacircle.
(A) Diagram depicting the strategy used to replace the first ~48 kb (Region 1) of the bcpAIOB-containing putative composite transposon (dark blue) with a cassette containing the gene that confers kanamycin resistance, nptII, and FRT-binding sites (pink). (B) PCR analyses to confirm removal of Region 1 in strain Reg1::nptII using primers P1 and P2 (shown as green arrows in panel A) which are close enough to generate a product only after deletion of the ~48 kb region. (C) PCR analyses of WT BtE264 and strain Reg1::nptII using primers P7 and P8, which would amplify two genes from within the Region 1 sequence. (D) Integration of a suicide plasmid (red arrowhead) within the Region 1 sequence of the mobilized element, followed by plasmid rescue studies, suggests that the mobilized megacircle inserted adjacent to the truncated composite transposon carrying Reg1::nptII mutation. (E) Comparison of the transformation efficiencies upon deletion of Region 1 in strains that are positive (WT) or deficient (ΔIS2β and Bt-Bp chimera) in the production of the megacircle. No DNA, black bars; DNA to introduce the Reg1::nptII mutation, red bars; DNA to introduce an nptII cassette outside of the putative composite transposon, blue bars; DNA to introduce an nptII cassette inside the putative composite transposon, green bars. Horizontal dashed line represents the average lowest limit of detection. P values were obtained using Mann-Whitney U test comparing mutant strains to WT when the same DNA (or no DNA) was added. Results are shown as mean +/- SEM of three independent experiments with three technical replicates each (n = 9). *P < 0.05; **P < 0.01.