Regulation of R1 Plasmid Transfer by H-NS, ArcA, TraJ, and DNA Sequence Elements

In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF₁₂A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated P_Y. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the P_Y promoter. To better understand the role of these proteins in P_Y promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJ_{R 1} (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1P_Y DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between -51 and -38 in the R1P_Y promoter region. Moreover, TraJ_{R 1} and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1P_Y regions proximal to the transcription start site. Our findings were corroborated by P_Y-lacZ reporter fusions with a series of site specific R1P_Y promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The P_Y promoter became activatable by F encoded TraJ whereas TraJ_{R 1} lost its activation function. The R1P_Y mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the -35 region is critical for P_Y regulation and function.