Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.

SignificanceKinesins are a superfamily of ATP-dependent motors important for many microtubule-based functions, including multiple roles in mitosis. Small-molecule inhibitors of mitotic kinesins disrupt cell division and are being developed as antimitotic therapies. We investigated the molecular mechanism of the multitasking human mitotic kinesin Kif18A and its inhibition by the small molecule BTB-1. We used cryo-electron microscopy to visualize nucleotide-dependent conformational changes in microtubule-bound Kif18A, and the conformation of microtubule-bound, BTB-1-bound Kif18A. We calculated a putative BTB-1–binding site and validated this site experimentally to reveal the BTB-1 inhibition mechanism. Our work points to a general mechanism of kinesin inhibition, with wide implications for a targeted blockade of these motors in both dividing and interphase cells.

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.