The multi-output incoherent feedforward loop constituted by the transcriptional regulators LasR and RsaL confers robustness to a subset of quorum sensing genes in Pseudomonas aeruginosa

Quorum sensing (QS) is an intercellular communication system which controls virulence-related phenotypes in the human pathogen Pseudomonas aeruginosa. LasR is the QS receptor protein which responds to the signal molecule N-(3-oxododecanoyl)homoserine lactone (3OC12-HSL) and promotes signal production by increasing the transcription of the 3OC12-HSL synthase gene, lasI. LasR also activates the expression of other genes, including rsaL, coding for the RsaL protein which acts as the transcriptional repressor of lasI. Direct gene activation and RsaL-mediated gene repression, both exerted by LasR on the expression of the output gene lasI, generate a regulatory network motif known as type 1 incoherent feedforward loop (IFFL-1) that governs 3OC12-HSL production. Beside lasI, RsaL directly represses a set of LasR-activated genes; hence, the IFFL-1 generated by LasR and RsaL is a multi-output IFFL-1. Here we demonstrate that the multi-output IFFL-1 constituted by LasR and RsaL confers robustness with respect to fluctuations in the levels of LasR to the phenotypes controlled by both these transcriptional regulators (e.g. 3OC12-HSL synthesis and pyocyanin production). On the contrary, other virulence-related phenotypes controlled by LasR but not by RsaL (e.g. elastase and proteases production) are sensitive to changes in LasR levels. As a whole, the multi output IFFL-1 generated by LasR and RsaL splits the QS regulon in two distinct sub-regulons with different robustness with respect to LasR fluctuations. This emerging regulatory property enhances phenotypic plasticity of P. aeruginosa, thus contributing to its adaptation to changing environments.