LasR, a Transcriptional Activator of Pseudomonas aeruginosa Virulence Genes, Functions as a Multimer

Abstract

The Pseudomonas aeruginosa LasR protein functions in concert with N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C 12 -HSL) to coordinate the expression of target genes, including many genes that encode virulence factors, with cell density. We used a LexA-based protein interaction assay to demonstrate that LasR forms multimers only when 3O-C 12 -HSL is present. A series of LasR molecules containing internal deletions or substitutions in single, conserved amino acid residues indicated that the N-terminal portion of LasR is required for multimerization. Studies performed with these mutant versions of LasR demonstrated that the ability of LasR to multimerize correlates with its ability to function as a transcriptional activator of lasI, a gene known to be tightly regulated by the LasR-3O-C 12 -HSL regulatory system. A LasR molecule that carries a C-terminal deletion can function as a dominant-negative mutant in P. aeruginosa, as shown by its ability to decrease expression of lasB, another LasR-3O-C 12 -HSL target gene. Taken together, our data strongly support the hypothesis that LasR functions as a multimer in vivo.An increasing number of bacterial species have been shown to utilize a cell density-dependent mechanism known as quorum sensing (QS) to regulate the expression of various genes (13, 28). The opportunistic pathogen Pseudomonas aeruginosa contains two QS systems, las and rhl, and the former is the best-characterized QS system in this organism (25,27). The las system is composed of the transcriptional activator LasR and its cognate autoinducer (AI) molecule, N-3-oxo-dodecanoyl-Lhomoserine lactone (3O-C 12 -HSL). Many of the target genes regulated by the LasR-3O-C 12 -HSL system or by RhlR and its cognate AI molecule, N-butyryl-L-homoserine lactone (C 4 -HSL), encode virulence factors, thereby implicating QS as an important constituent of P. aeruginosa pathogenesis.LasR and RhlR belong to the LuxR protein family, which was named after the regulator of bioluminescence (lux) in Vibrio fischeri (36). Members of this family act as transcriptional activators and exhibit homology with LuxR in two distinct regions of the protein. The first region appears to be required for AI binding and is located in the N-terminal portion of the protein. The second region resides within the carboxyl-terminal portion of the protein and appears to characterize a DNA-binding domain (DBD) containing a helix-turnhelix motif (36).The current paradigm for the activation of gene expression in QS suggests that the protein-AI complex interacts with a specific DNA element and, perhaps through contacts with RNA polymerase, stimulates target gene expression (35,36). The binding of AI by the activator protein is believed to result in conformational changes that allow formation of a functional form of the protein. Several studies have indicated that proteins belonging to this family interact with DNA (16, 17, 34-36, 42, 44), and the existence of dyad symmetry in the proposed DNA element suggests that members of the LuxR family of proteins might ...

Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR

“…We observed a single peak with a radius of 3.15 nm (data not shown) corresponding to a molecular mass of 49.3 kDa. This finding suggested that purified LasR-3OC12-HSL exists as a dimer in solution, consistent with a genetic study showing that LasR functions as a multimer in vivo (30).…”

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…Both biofilm formation and pyocyanin production, two best known QS phenomena in P. aeruginosa [27], were not significantly changed when the CRISPR-Cas system was deleted (Supplementary information, Figure S8), suggesting that CRISPR-Cas system does not impact on host inflammatory responses through modulating the regulatory activity of LasR in QS. As all of the above-mentioned data were collected using bacteria from stationary phase (OD 600 ~2.0), we repeated certain key experiments using the OD 600 ~0.5 cultures (log phase) of PA14 strains ( Figure 6D-6F).…”

“…As LasR is the critical QS regulator [27], we compared the QS phenomenon between PA14 WT and the mutants. Both biofilm formation and pyocyanin production, two best known QS phenomena in P. aeruginosa [27], were not significantly changed when the CRISPR-Cas system was deleted (Supplementary information, Figure S8), suggesting that CRISPR-Cas system does not impact on host inflammatory responses through modulating the regulatory activity of LasR in QS.…”

QsIA disrupts LasR dimerization in antiactivation of bacterial quorum sensing

“…Previous studies indicate that the binding of the autoinducer 3-oxo-C12-HSL stabilizes LasR and promotes its dimerization, thereby enabling the resultant homodimeic LasR-autoinducer complex to bind to its target DNA promoter and activate gene transcription (6,13,22,24,25). We have shown previously that QslA can associate with LasR and thus disturb its target DNA-binding ability (22).…”