ABSTRACTPseudomonas aeruginosais a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection,P. aeruginosacoordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signalN-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and thePseudomonasquinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription ofpqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in thepqsRpromoter region and that it does not influence the transcription of the divergently transcribed gene,nadA. Using DNA affinity chromatography, we identified additional proteins that interact with thepqsR-nadAintergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with thepqsRpromoter and that CysB repressespqsRtranscription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation ofpqsRtranscription by LasR. However, as seen with other CysB-regulated genes,pqsRexpression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production byP. aeruginosa.IMPORTANCEThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of theP. aeruginosacell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation ofpqsR. We identified CysB as a novel regulator ofpqsRand PQS production, but, unlike other CysB-controlled genes, it does not appear to regulatepqsRin response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator inP. aeruginosa.