Plasmid r1 conjugative DNA processing is regulated at the coupling protein interface.
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.
Mihajlovic
Sanja
S
University of Graz, Institute of Molecular Biosciences, Humboldtstrasse 50, A-8010 Graz, Austria.
Lang
Silvia
S
Sut
Marta V
MV
Strohmaier
Heimo
H
Gruber
Christian J
CJ
Koraimann
Günther
G
Cabezón
Elena
E
Moncalián
Gabriel
G
de la Cruz
Fernando
F
Zechner
Ellen L
EL
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
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