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 aeruginosaElectronic supplementary information (ESI) available. See DOI: 10.1039/c7mb00040e

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 (3OC 12 -HSL) and promotes signal production by increasing the transcription of the 3OC 12 -HSL synthase gene, lasI . LasR also activates the expression of other genes, including rsaL , coding for the RsaL protein which acts as a 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 the type 1 incoherent feedforward loop (IFFL-1) that governs 3OC 12 -HSL production. In addition to 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. 3OC 12 -HSL synthesis and pyocyanin production). In contrast, other virulence-related phenotypes controlled by LasR but not by RsaL ( e.g. elastase and protease production) are sensitive to changes in LasR levels. Overall, the multi-output IFFL-1 generated by LasR and RsaL splits the QS regulon into two distinct sub-regulons with different robustness with respect to LasR fluctuations. This emerging regulatory property enhances the phenotypic plasticity of P. aeruginosa , thus contributing to its adaptation to changing environments. The las multi-output IFFL-1 splits the QS regulon into two distinct sub-regulons with different robustness with respect to LasR fluctuations.