BrlR from Pseudomonas aeruginosa is a c‐di‐GMP‐responsive transcription factor

Abstract

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators that contributes to the high-level drug tolerance of Pseudomonas aeruginosa biofilms. While MerR regulators are known to activate both the expression of multidrug efflux pump genes and their own transcription upon inducer-binding, little is known about BrlR activation. We demonstrate using promoter reporter strains, in vivo and in vitro DNA-binding assays combined with 5’RACE, that BrlR binds to its own promoter, likely via a MerR-like palindromic sequence. Unlike known MerR multidrug transport activators, BrlR and brlR expression are not activated by multidrug transporter substrates. Instead, BrlR-DNA binding was enhanced by the secondary messenger c-di-GMP. In addition to enhanced BrlR-DNA binding, c-di-GMP levels contributed to PbrlR promoter activity in initial attached cells with elevated c-di-GMP levels correlating with increased expression of brlR. While not harboring amino acid motifs resembling previously defined c-di-GMP-binding domains, BrlR was found to bind c-di-GMP in vitro at a ratio of one c-di-GMP per two BrlR. Crosslinking assays confirmed dimer formation to be enhanced in the presence of elevated c-di-GMP levels. Our findings demonstrate BrlR to be an unusual MerR-family member in that BrlR function and expression require the secondary messenger c-di-GMP.

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

“…While most early studies documented c-di-GMP regulation at the posttranslational level, recent characterization of the DNA-binding proteins VpsT, BldD, Bcam1349, BrlR, CLP, FleQ/FlrA, LtmA, MrkH, and VpsR (33,57,61,62,(70)(71)(72)(73)(74)(75) provided clear examples of c-di-GMP involvement in transcriptional regulation. Remarkably, while most of these transcriptional regulators bind DNA via standard helix-turn-helix domains, MrkH combines its c-di-GMP-binding PilZ domain with a previously unknown type of the DNA-binding domain, which is related to PilZN/YcgR and PilZNR/YcgR_2 domains (see Fig.…”

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

“…LtmA, an apparently new class of regulator from Mycobacterium smegmatis, binds c-di-GMP to positively regulate the expression of lipid transport and metabolism genes (47). Finally, BrlR, a MerR family member from P. aeruginosa, was recently shown to bind c-di-GMP to control gene expression (48).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…The inactivation of SagS correlates with a reduced level of c-di-GMP, and thus, c-di-GMP is positively regulated by SagS (823). c-di-GMP further positively affects the production of BrlR and also enhances the binding of BrlR to the promoters of BrlR target genes (821,823). Together, SagS, BrlR, and c-di-GMP form an important signaling network related to the susceptibility-resistance switch and are all required for elevated expression in biofilm cells of the MexAB-OprM and MexEFOprN pumps, which contribute to resistance in both biofilm and planktonic cells.…”

“…This is attributed to the regulatory activation of mexAB-oprM and mexEF-oprN by BrlR's binding to the promoter regions of these efflux operons [819].) Both BrlR production and BrlR-DNA binding are stimulated by the secondary messenger cyclic di-GMP (c-di-GMP) (821). Since MexAB-OprM does not accommodate aminoglycosides unless bacteria are cultured in low-ionic-strength medium (164), the reported hypersusceptibility of PAO1⌬brlR biofilms to tobramycin and kanamycin might be interpreted as the result of OprM being in limiting amounts to form a functional MexXY/OprM system able to extrude these molecules (434).…”