AFM-based force spectroscopy unravels the stepwise-formation of a DNA transposition complex driving multi-drug resistance dissemination

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. So far, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds to a single transposon end and then undergoes a structural transition that enables it to bind the second end co-operatively and to become activated for transposition catalysis. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.