Conjugation is a crucial pathway for horizontal gene transfer, significantly contributing to genome diversification and the spread of antimicrobial resistance (AMR) genes. Central to this process is the Type IV secretion system (T4SS), a complex molecular apparatus that facilitates unidirectional DNA transfer between bacterial cells. This sophisticated system is formed by at least 12 different proteins that assemble into a cell envelope-spanning complex, an extracellular pilus, and cytosolic nucleoprotein complex. Within this system, the multi-domain protein TraI plays a central role in DNA recognition, processing, and translocation. Despite advances in our understanding of T4SS, the precise structural dynamics of DNA translocation – particularly how the TraI-DNA complex engages and progresses through the secretion channel – remain incompletely characterized, impeding the development of new strategies to halt the spread of AMR.
Here, we focus on TraI of the F-like conjugative plasmid pED208 and develop tools to enable structural and functional characterization of T4SS during active DNA transfer. Specifically, translational fusion of TraI to the highly stable synthetic protein TOP7 were generated as a non-translocatable folding susbtrates to stall the system in its active transfer state. Functionality of the engineered stalling substrates was established using fluorescent reporter tools that confirm TraI-DNA binding, substrate recognition and stalling of the translocation process. Future experiments combining fluorescence microscopy and cryo electron tomography will facilitate investigation of T4SS structural states associated with active DNA transfer.
By enabling visualization of conjugation in action, this work will provide new insights into the molecular mechanisms underlying bacterial DNA transfer and establish a framework for identifying structural features critical for conjugation. Ultimately, this knowledge may inform strategies to interfere with bacterial conjugation and limit dissemination of AMR genes.