Inactivation of the dimeric Rap(pLS20) anti-repressor of the conjugation operon is mediated by peptide-induced tetramerization

Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. Rap(pLS20) is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by Rco(pLS20) in the absence of the mature pLS20 signaling peptide Phr*(pLS20). We report here an in-depth structural study of apo and Phr*(pLS20)-bound states of Rap(pLS20) at various levels of atomic detail. We show that apo-Rap(pLS20) is dimeric and that Phr*(pLS20)-bound Rap forms NTD-mediated tetramers. In addition, we show that Rap(pLS20) binds Rco(pLS20) directly in the absence of Phr*(pLS20) and that addition of Phr*(pLS20) releases Rco(pLS20) from Rap(pLS20). This allows Rco(pLS20) to bind the promotor region of crucial conjugation genes blocking their expression.