ABSTRACT
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.
Subject(s)
Agrobacterium tumefaciens/ultrastructure , Macromolecular Substances/isolation & purification , Macromolecular Substances/ultrastructure , Type IV Secretion Systems/isolation & purification , Type IV Secretion Systems/ultrastructure , Agrobacterium tumefaciens/genetics , Conjugation, Genetic , Microscopy, Electron, Transmission , Protein Interaction MapsABSTRACT
Conjugation, the process by which plasmid DNA is transferred from one bacterium to another, is mediated by type IV secretion systems (T4SSs). T4SSs are versatile systems that can transport not only DNA, but also toxins and effector proteins. Conjugative T4SSs comprise 12 proteins named VirB1-11 and VirD4 that assemble into a large membrane-spanning exporting machine. Before being transported, the DNA substrate is first processed on the cytoplasmic side by a complex called the relaxosome. The substrate is then targeted to the T4SS for export into a recipient cell. In this review, we describe the recent progress made in the structural biology of both the relaxosome and the T4SS.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Conjugation, Genetic , Gram-Negative Bacteria/genetics , Type IV Secretion Systems/chemistry , Type IV Secretion Systems/physiology , DNA, Bacterial/metabolism , Gram-Negative Bacteria/metabolism , Models, Molecular , Plasmids , Type IV Secretion Systems/geneticsABSTRACT
Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized 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.
Subject(s)
Bacterial Secretion Systems , Escherichia coli/chemistry , Escherichia coli/ultrastructure , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Bacterial Secretion Systems/genetics , Cell Membrane/metabolism , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/ultrastructure , Microscopy, Electron , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructureABSTRACT
Proteins containing selenocysteine are found in members of all three domains of life, Bacteria, Eukarya and Archaea. A dedicated tRNA (tRNA(sec)) serves as a scaffold for selenocysteine synthesis. However, sequence and secondary structures differ in tRNA(sec) from the different domains. An Escherichia coli strain lacking the gene for tRNA(sec) could only be complemented with the homologue from Methanococcus maripaludis when a single base in the anticodon loop was exchanged demonstrating that this base is a crucial determinant for archaeal tRNA(sec) to function in E. coli. Complementation in trans of M. maripaludis JJ mutants lacking tRNA(sec) , O-phosphoseryl-tRNA(sec) kinase or O-phosphoseryl-tRNA(sec) :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.
