Strains and plasmids used in this study.

<div><p><i>Pseudomonas aeruginosa</i> is an opportunist bacterium that causes acute and chronic infections. During acute infections, the type III secretion system (T3SS) plays a pivotal role in allowing the bacteria to translocate effectors such as ExoS, ExoT, and ExoY into host cells for colonization. Previous research on the involvement of quorum sensing systems Las and Rhl in controlling the T3SS gene expression produced ambiguous results. In this study, we determined the role of the Las and Rhl systems and the PqsE protein on T3SS expression. Our results show that in the wild-type PAO1 strain, the deletion of <i>lasR</i> or <i>pqsE</i> do not affect the secretion of ExoS. However, <i>rhlI</i> inactivation increases the expression of T3SS genes. In contrast to the <i>rhlI</i> deletion, <i>rhlR</i> inactivation decreases both T3SS genes expression and ExoS secreted protein levels, and this phenotype is restored when this mutant is complemented with the <i>exsA</i> gene, which codes for the master regulator of the T3SS. Additionally, cytotoxicity is affected in the <i>rhlR</i> mutant strain compared with its PAO1 parental strain. Overall, our results indicate that neither the Las system nor PqsE are involved in regulating the T3SS. Moreover, the Rhl system components have opposite effects, RhlI participates in negatively controlling the T3SS expression, while RhlR does it in a positive way, and this regulation is independent of C4 or PqsE. Finally, we show that <i>rhlR</i>, <i>rhlI</i>, or <i>pqsE</i> inactivation abolished pyocyanin production in T3SS-induction conditions. The ability of RhlR to act as a positive T3SS regulator in the absence of its cognate autoinducer and PqsE shows that it is a versatile regulator that controls different virulence traits allowing <i>P</i>. <i>aeruginosa</i> to compete for a niche.</p></div>