title: Interaction between the co-inherited TraG coupling protein and the TraJ membrane-associated protein of the H-plasmid conjugative DNA transfer system resembles chromosomal DNA translocases.
abstract: Bacterial conjugation is a DNA transfer event that requires three plasmid-encoded multi-protein complexes: the membrane-spanning mating pair formation (Mpf) complex, the cytoplasmic nucleoprotein relaxosome complex, and a homo-multimeric coupling protein that links the Mpf and relaxosome at the cytoplasmic membrane. Bacterial two-hybrid (BTH) technology and immunoprecipitation were used to demonstrate an interaction between the IncH plasmid-encoded transfer protein TraJ and the coupling protein TraG. TraJ is essential for conjugative transfer but is not required for the formation of the conjugative pilus, and is therefore not regarded as an Mpf component. Fractionation studies indicated that TraJ shared a similar cellular domain to that of TraG at the cellular membrane. Protein blast analyses have previously identified TraJ homologues encoded in a multitude of plasmid and chromosomal genomes that were also found to encode an adjacent TraG homologue, thus indicating co-inheritance. BTH analysis of these TraJ and cognate TraG homologues demonstrated conservation of the TraJ-TraG interaction. Additional occurrences of the traJ-traG module were also detected in genomic sequence data throughout the Proteobacteria, and phylogenetic comparison of these IncH-like TraG proteins with the coupling proteins encoded by other conjugative transfer systems (including IncP, IncW and IncF) that lack TraJ homologues indicated that the H-like coupling proteins were distinct. Accordingly, the IncP, IncW and IncF coupling proteins were unable to interact with TraJ, but were able to interact with IncH plasmid-encoded TrhB, an Mpf component known to complex with its cognate coupling protein TraG. The divergence of the IncH-type coupling proteins may partly be due to the requirement of TraJ interaction, and notably, TraG and TraJ cumulatively represent the domain architecture of the known translocase family FtsK/SpoIIIE. It is proposed that TraJ is a functional part of the IncH-type coupling protein complex required for translocation of DNA through the cytoplasmic membrane.
authors.affiliation: Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: James E
authors.initials: JE
authors.lastname: Gunton
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Gilmour
authors.affiliation: Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Kelly P
authors.initials: KP
authors.lastname: Baptista
authors.affiliation: Department of Medical Microbiology, Stanford University, CA 94305, USA.
authors.forename: Trevor D
authors.initials: TD
authors.lastname: Lawley
authors.affiliation: Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Taylor
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000201:enzymology; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C073791:TraG protein, E coli; C488999:TraJ protein, E coli