Cooperative Function of TraJ and ArcA in Regulating the F Plasmid <i>tra</i> Operon

Abstract

The F plasmid <i>tra</i> operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the <i>tra</i> operon promoter P_Y, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that P_Y is inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused by <i>tra</i> expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to P_Y regulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the P_Y regulatory region defined a 22-bp tandem repeat sequence (from -76 to -55 of P_Y) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from -54 to -34). Deletion analysis of the P_Y promoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of -103 are required by silencers including H-NS for P_Y silencing, whereas sequences downstream of -77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract P_Y silencers but also to directly activate P_Y in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during P_Y activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.<b>IMPORTANCE</b> Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The <i>tra</i> operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of <i>tra</i> operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major <i>tra</i> promoter P_Y The interaction of TraJ and ArcA with the <i>tra</i> operon not only relieves P_Y from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.