title: Plasmid r1 conjugative DNA processing is regulated at the coupling protein interface.
abstract: Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDDeltaN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (DeltaL(k) = -4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDDeltaN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.
authors.affiliation: University of Graz, Institute of Molecular Biosciences, Humboldtstrasse 50, A-8010 Graz, Austria.
authors.forename: Sanja
authors.initials: S
authors.lastname: Mihajlovic
authors.forename: Silvia
authors.initials: S
authors.lastname: Lang
authors.forename: Marta V
authors.initials: MV
authors.lastname: Sut
authors.forename: Heimo
authors.initials: H
authors.lastname: Strohmaier
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Gruber
authors.forename: Günther
authors.initials: G
authors.lastname: Koraimann
authors.forename: Elena
authors.initials: E
authors.lastname: Cabezón
authors.forename: Gabriel
authors.initials: G
authors.lastname: Moncalián
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Ellen L
authors.initials: EL
authors.lastname: Zechner
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C064957:traD protein, E coli; C027093:TraI protein, E coli; D004265:DNA Helicases