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

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.

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:selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a selenium-independent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the 'history' of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.