Structure of a type IV secretion system

Abstract

Bacterial type IV secretion (T4S) systems translocate virulence factors into eukaryotic cells 1,2 , distribute genetic material between bacteria, and have shown potential as a tool for the genetic modification of human cells 3 . Given the complex choreography of the substrate through the secretion apparatus 4 , the molecular mechanism of the T4S system has proven difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy (EM) to reconstruct the T4S system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form a ~3 megadalton (MDa) nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex 1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of twelve VirB4 ATPase subunits organised as side by side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems 1,4-6 .The canonical T4S system comprises 12 proteins, VirB1-11 and VirD4, and forms a large macromolecular complex that spans the cell envelope of Gram-negative bacteria 2 . The hub protein VirB10 inserts into both the inner and outer membranes and spans the entire width of the periplasm. It is decorated by VirB7 and VirB9 in a 1:1:1 ratio to form a C14 symmetrised outer membrane pore termed the core complex 7 . The architecture and relative † Correspondence and requests for materials should be addressed to GW

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

“…Salmonella motors and C. jejuni ΔpflA, ΔpflB, ΔflgP, and ΔflgQ exhibited flexibility between the top and bottom parts of the motor. In these cases individual averages were constructed for the top and bottom segments and ultimately were merged into a composite structure in analogy to recent single-particle studies (62).…”

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

“…All other bands observed in the SDS–PAGE analysis of the complex purified from TrwB /VirD4 ‐producing cells are also present in the T4SS 3‐10 complex. This includes OmpC and OmpA, contaminants previously proven to be minor (Low et al , 2014). Thus, the complex purified from TrwB /VirD4 ‐producing cells is the T4SS 3‐10 complex to which VirD4 is bound.…”

“…The labelled sample of the complex was analysed using SDS–PAGE, and the fluorescence intensity of the bands was quantified. The TrwE /VirB10 protein as a part of the TrwH /VirB7 ‐TrwF /VirB9 ‐TrwE /VirB10 core complex alone or of the T4SS 3‐10 complex is known to be present in 14 copies (Fronzes et al , 2009b; Low et al , 2014). Therefore, the fluorescence signal quantified from the TrwE /VirB10 band was used as a reference to determine the stoichiometry of the other components.…”

“…This is the issue we set out to address in this study. In order to achieve this, we designed functional expression systems that resulted in TrwB /VirD4 forming a stable complex with the previously investigated T4SS 3‐10 complex (Low et al , 2014). This interaction is primarily mediated by the core OMC complex as deletion of TrwM /VirB3 or TrwK /VirB4 results in the formation of a complex between TrwB /VirD4 and the core OMC complex alone.…”

“…The inner‐layer (I‐layer) is assembled from the N‐terminal parts of VirB9 and VirB10 with the very N‐terminal part of VirB10 extending into the cytosol and inserting a transmembrane helix into the inner membrane. Recently, the structure of a larger complex containing eight VirB components including the VirB3‐10 proteins (a complex referred to thereafter as T4SS 3‐10 ) was solved by negative stain electron microscopy (NS‐EM), revealing the structure of the inner membrane part of a T4S system or inner membrane complex (IMC) composed of VirB3‐6 and VirB8 (Low et al , 2014). The most prominent features of the IMC are two barrel‐like structures which are each divided into three layers: the lower, middle and upper tiers.…”