title: Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB.
abstract: Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.
authors.affiliation: Department of Medical Microbiology, 1-28 Medical Sciences Building, University of Alberta, Edmonton, Canada T6G 2R3.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Gilmour
authors.forename: James E
authors.initials: JE
authors.lastname: Gunton
authors.forename: Trevor D
authors.initials: TD
authors.lastname: Lawley
authors.forename: Diane E
authors.initials: DE
authors.lastname: Taylor
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C073791:TraG protein, E coli; C482104:TrhB protein, E coli