IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes

<jats:p>Relaxosomes are specific nucleoprotein structures involved in DNA‐processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (<jats:italic>oriT)</jats:italic> DNA plus three proteins TrwC relaxase, TrwA <jats:italic>nic</jats:italic>‐cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (<jats:italic>ihfA</jats:italic> and <jats:italic>ihfB</jats:italic>) in R388 <jats:italic>oriT</jats:italic>, as shown by gel retardation and DNase I footprinting analysis. IHF binding <jats:italic>in vitro</jats:italic> was found to inhibit <jats:italic>nic</jats:italic>‐cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF<jats:sup>−</jats:sup> and IHF<jats:sup>+</jats:sup> donor strains. In contrast, examination of plasmid DNA obtained from IHF<jats:sup>−</jats:sup> strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF<jats:sup>+</jats:sup> strains. Thus, IHF could have an inhibitory role in the <jats:italic>nic</jats:italic>‐cleavage reaction <jats:italic>in vivo</jats:italic>. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from <jats:italic>oriT</jats:italic>.</jats:p>