CysB Negatively Affects the Transcription of pqsR and Pseudomonas Quinolone Signal Production in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is 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. aeruginosa coordinates 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 signal N-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and the Pseudomonas quinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription of pqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in the pqsR promoter 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 the pqsR-nadA intergenic 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 the pqsR promoter and that CysB represses pqsR transcription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation of pqsR transcription by LasR. However, as seen with other CysB-regulated genes, pqsR expression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production by P. aeruginosa. IMPORTANCEThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of the P. aeruginosa cellto-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 of pqsR. We identified CysB as a novel regulator of pqsR and PQS production, but, unlike other CysB-controlled genes, it does not appear to regulate pqsR in response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator in P. aeruginosa. Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment, due in part to its metabolic versatility that allows it to obtain the nutrients needed for growth from a wide variety of compounds (1). P. aeruginosa is also an opportunistic pathogen that can infect plants, nematodes, insects, and mammals (2-5). With regard to humans, it can cause a wide range of diseases and is a major cause of health care-associated illnesses and devastating chronic infections in cystic fibrosis patients (6, 7). In order to thrive under such a diverse range of conditions, P. aeruginosa utilizes complex regulatory networks to control gene expression. These networks include global regulatory systems that can direct l...

Distal and proximal promoters co‐regulate pqsR expression in Pseudomonas aeruginosa

“…This finding suggested that transcriptional initiation at the TS3/4 promoter site was very efficient, but it was unclear why we observed such a large difference in activity when lacZ was fused within the pqsR coding region. Finally, we examined the role of LasR in the activation of both the full‐length pqsR'‐lacZ fusion and the pqsR TS3/4‐ lacZ fusion by altering base pairs in the LasR binding site on each to prevent LasR from interacting with the pqsR promoter region (Farrow et al ., ). These mutations caused the β‐gal activity produced by each of the lacZ fusions to be greatly reduced (Fig.…”

“…Data are presented in Miller units as the mean 6 SD of results from duplicate assays from at least three separate experiments. promoter region (Farrow et al, 2015). These mutations caused the b-gal activity produced by each of the lacZ fusions to be greatly reduced (Fig.…”

“…C). Interestingly, we have previously shown that purified CysB interacted with two sites in the pqsR promoter region: it bound to one fragment that contained the LasR binding site, and also with lower affinity to a fragment containing the TS1 and TS2 (Farrow et al ., ). Together these findings suggest that CysB can positively affect pqsR transcription at a proximal promoter site, although it is uncertain whether this form of regulation occurs under normal physiological conditions.…”

“…LasR activates pqsR transcription at a distal promoter site Previously we found that LasR only interacts with a single binding site in the pqsR promoter region centered at 278 bp upstream from the pqsR transcriptional start site 1 (TS1), and that this binding site was important for the expression of pqsR (Farrow et al, 2015). However, the mechanism used by LasR to activate transcription from this distal binding site was unclear.…”

“…The pqsR coding sequence is separated from the divergently transcribed nadA gene by 747 bp, and two transcriptional start sites were identified at 2190 and 2278 bp relative to the pqsR translational start site (Wade et al, 2005). LasR positively regulates pqsR transcription from a binding site that is distant from these start sites (Xiao et al, 2006b;Farrow et al, 2015). Additionally, upstream from pqsR is a small open reading frame (uof), and the translation of uof appears to be linked to the translation of pqsR, providing a point for post-transcriptional control of PqsR expression (Sonnleitner et al, 2011).…”