Cooperative Function of TraJ and ArcA in Regulating the F Plasmid tra Operon

Abstract

The F plasmid tra operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the tra operon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PY 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 tra expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PY 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 PY regulatory region defined a 22-bp tandem repeat sequence (from −76 to −55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from −54 to −34). Deletion analysis of the PY 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 PY silencing, whereas sequences downstream of −77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PY silencers but also to directly activate PY in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PY activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation. IMPORTANCE Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The tra operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of tra operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major tra promoter PY. The interaction of TraJ and ArcA with the tra operon not only relieves PY from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

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

“…Unspecific DNA binding was also observed in the band shift experiments and is possibly due to our in vitro conditions which do not reflect the true in vivo situation. Weak DNA binding and low specificity have also been observed for TraJ F binding to the FP Y promoter; in addition, the authors of that study suggested a cooperative binding mode without a direct protein-protein interaction (Lu et al, 2018). Our findings that ArcA-P binding to R1P Y DNA somehow enhanced TraJ R1 binding to jbs containing DNA is consistent with this notion.…”

“…It has similarly been shown that the SsrB response regulator of S. enterica can replace H-NS from type III effector gene promoters in SPI-2 required for intracellular growth and maintenance of this pathogen (Walthers et al, 2011). A cooperative activity of TraJ F and ArcA has also recently been proposed to mediate activation of the FP Y promoter which is also silenced by H-NS (Will and Frost, 2006;Rodriguez-Maillard et al, 2010;Lu et al, 2018). The results of our promoter mutagenesis studies fully supported the idea that counter-silencing in the R1P Y promoter requires ArcA since when only two bases in the second ArcA box hexamer were exchanged (from TGTTAA to TCATAA), together with a lower basal activity, induction of the R1P Y promoter was virtually abolished.…”

“…The key plasmid encoded activator that has to escape negative control is TraJ, a plasmid encoded protein. As schematically depicted in Figure 1A, TraJ and chromosomally encoded ArcA proteins are absolutely required to overcome heat-stable nucleoid structuring (H-NS) protein silencing and to activate transcription of DNA transfer genes from the main transfer operon promoter P Y (Will and Frost, 2006;Wagner et al, 2013;Lu et al, 2018).…”

Plasmid Transfer by Conjugation in Gram-Negative Bacteria: From the Cellular to the Community Level

“…The H-NS copy number per cell varies during growth [ 20 ], thus rendering the F plasmid transfer rate growth phase-dependent, i.e., maximum in the exponential phase, reduced in the mid-exponential phase, and mostly abolished in the stationary phase [ 21 , 22 ]. However, during the exponential phase, H-NS repression activity is itself counteracted by the cooperative binding of TraJ and the host protein ArcA (aerobic respiration control of anoxic redox control) to the P Y promoter [ 23 ]. In the case of the virulence plasmid pSLT of Salmonella enterica , H-NS repression activity also reportedly depends on Dam (DNA adenine methylase) methylation of the DNA [ 24 ].…”

Bacteria DNA Conjugation: from The Cellular to The Community Level

“…H-NS copy number per cell varies during growth [20], thus rendering F plasmid transfer rate growth-phase-dependent, i.e., maximum in exponential, reduced in mid-exponential and mostly abolished in stationary phase [21,22]. Yet, during the exponential phase, H-NS repression activity is itself counteracted by the cooperative binding of TraJ and the host protein ArcA (Aerobic respiration control of anoxic redox control) to the PY promoter [23]. In the case of the virulence plasmid pSLT of Salmonella enterica, H-NS repression activity was also reported to depend on Dam (DNA adenine methylase) methylation of the DNA [24].…”