Translocation through the conjugative Type 4 secretion system requires unfolding of its protein substrate

Bacterial conjugation, a mechanism of horizontal gene transfer, is the major means by which antibiotic resistance spreads among bacteria (1, 2). Conjugative plasmids are transferred from one bacterium to another through a type IV secretion system (T4SS) in a form of single-stranded DNA covalently attached to a protein called relaxase. The relaxase is fully functional both in a donor cell (prior to conjugation) and recipient cell (after conjugation). Here we demonstrate that the protein substrate has to unfold for efficient translocation through the conjugative T4SS. Furthermore, we present various relaxase modifications that preserve the function of the relaxase but block substrate translocation. This study brings us a step closer to deciphering the complete mechanism of T4SS substrate translocation, vital for development of new therapies against multidrug-resistant pathogenic bacteria. Importance Conjugation is the principal means by which antibiotic resistance genes spread from one bacterium to another (1, 2). During conjugation, a covalent complex of single-stranded DNA and a protein termed relaxase is transported by a type IV secretion system. To date, it is not known whether the relaxase requires unfolding prior to transport. In this report, we use functional assays to monitor the transport of relaxase wild-type and variants containing unfolding-resistant domains and show that these domains reduce conjugation and protein transport dramatically. Mutations that lower the free energy of unfolding in these domains do not block translocation and can even promote it. We thus conclude that the unfolding of the protein substrate is required during transport

LIS1 Clamps Dynein to the Microtubule

Cytoplasmic dynein is a motor essential for numerous mechanical processes in eukaryotic cells. How its activity is regulated is largely unknown. By using a combination of approaches including single-molecule biophysics and electron microscopy, Huang et al. in this issue uncover the regulatory mechanism by which LIS1 controls the activity of cytoplasmic dynein

Recent advances in the structural and molecular biology of type IV secretion systems

Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1 MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of ∼3 MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations

Secretion systems in Gram-negative bacteria: structural and mechanistic insights

Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies