Induction of Plasmid Conjugation in Bacillus subtilis Is Bistable and Driven by a Direct Interaction of a Rap/Phr Quorum-sensing System with a Master Repressor

Conjugation of plasmid pLS20 from Bacillus subtilis is limited to a time window between early and late exponential growth. Genetic evidence has suggested that pLS20-encoded protein Rco(LS20) represses expression of a large conjugation operon, whereas Rap protein Rap(LS20) relieves repression. We show that Rap(LS20) is a true antirepressor protein that forms dimers in vivo and in vitro and that it directly binds to the repressor protein Rco(LS20) in a 1: 1 stoichiometry. We provide evidence that Rap(LS20) binds to the helix-turn-helix-containing domain of Rco(LS20) in vivo, probably obstructing DNA binding of Rco(LS20), as seen in competitive DNA binding experiments. The activity of Rap(LS20) in turn is counteracted by the addition of the cognate Phr(LS20) peptide, which directly binds to the Rap protein and presumably induces a conformational change of the antirepressor. Thus, a Rap protein acts directly as an antirepressor protein during regulation of plasmid conjugation, turning on conjugation, and is counteracted by the Phr(LS20) peptide, which, by analogy to known Rap/Phr systems, is secreted and taken back up into the cells, mediating cell density-driven regulation. Finally, we show that this switchlike process establishes a population heterogeneity, where up to 30% of the cells induce transcription of the conjugation operon.