Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor Rap(pLS20)

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, P(c), is by default repressed by a regulator Rco(pLS20) involving DNA looping. A plasmid-encoded signalling peptide, Phr*(pLS20), inactivates the anti-repressor of Rco(pLS20), named Rap(pLS20), which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two Rco(pLS20) tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that Rap(pLS20) is sufficient for activating conjugation in vivo. We also show that Rap(pLS20) actively detaches Rco(pLS20) from DNA by preferentially acting on the Rco(pLS20) molecules involved in DNA looping, resulting in sequestration but not inactivation of Rco(pLS20). Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.