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Abstract

The spread of antibiotic resistance genes (ARGs) has become one of the biggest health care challenges in the last decades. Conjugative transposons (CTns) are a major class of mobile genetic elements that can transfer antibiotic resistance genes between bacterial genomes. For CTn transposition, DNA cleavage and joining reactions are catalyzed by a transposon-encoded integrase enzyme (Int). However, transposition doesn’t involve only Int but requires rather complex machinery, which employs multiple CTn- and host-encoded factors assembled in distinct higher order protein-DNA complexes. These assemblies act as hubs to regulate recombination both spatially and temporally. Despite available biochemical and structural data about some CTn Int proteins, the coordination and regulation of the recombination reaction by higher order complex formation are not well understood. During my doctoral studies, I aimed to decipher the structural and functional principles of higher order nucleoprotein assemblies in the transposition of the GISul2 element, a wide host range CTn that propagates diverse antibiotic resistance genes in pathogenic Gram-negative bacteria. In the context of this work, several structures of transposon excision complexes formed at different steps of the reaction were determined using Cryo-EM, which revealed distinct molecular assemblies. First, the structure of the right transposon end (RE) complex showed that RE DNA is bent by integration host factor (IHF) to ~160°, which allows IntGISul2 to bridge its arm and core DNA sites. The formation of this complex strictly requires the presence of the host-encoded accessory protein IHF, indicating strong coordination between the host cell state and CTn mobilization. In the second part, I present the structural characterization of the left transposon end (LE) complex. The cryo-EM map revealed that seven excisionase (Xis) molecules bend the LE DNA to form a loop that is tethered by integrases. LE complex formation depends on DNA bending by the transposon-encoded Xis protein, a directionality factor of recombination, which is required for excision but inhibits integration. In the third part of the thesis, I describe structural insights into how right and left transposon ends come together to form a synaptic complex during transposon excision. This synaptic complex was captured using “suicide” DNA substrates, which can stall the recombination reaction after cleavage of the first DNA strand pair. Unexpectedly, this approach resulted in the formation of two different synaptic complexes, one including RE and LE, and the other one containing two RE molecules, which together elucidate the assembly process and catalytic activation of the native synaptic excision complex. Together our results shed light on the principles of how the CTn integrase cooperates with accessory DNA bending proteins to coordinate active nucleoprotein complex assembly during excision and integration. These insights offer new knowledge about the mechanisms of antibiotic resistance spreading in Gram-negative pathogens. I hope that the better understanding of the CTn movement will help develop new strategies for tackling antibiotic resistance spread in the future.