Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

FIG. 1.

Neither LasR nor RhlR binds the pqsA promoter. (A) Radiolabeled DNA containing the pqsA promoter region was added to a LasR-containing cell lysate that was prepared in the absence (lanes 1 to 5) or presence (lanes 6 to 10) of 10 μM 3-oxo-C₁₂-HSL (C₁₂). (B) Radiolabeled DNA from the pqsA promoter region was added to an RhlR-containing cell lysate prepared in the absence (lanes 1 to 5) or presence (lanes 6 to 10) of 10 μM C₄-HSL (C₄). (C) Radiolabeled DNA from the lasB promoter region was added to a LasR-containing cell lysate prepared in the presence of 10 μM 3-oxo-C₁₂-HSL. (D) Radiolabeled DNA from the rhlA promoter region was added to an RhlR-containing cell lysate prepared in the absence of C₄-HSL. The total protein concentration of each lysate was determined, and the following amounts of protein were added per reaction: lanes 1 and 6, 0 μg; lanes 2 and 7, 10 μg; lanes 3 and 8, 20 μg; lanes 4 and 9, 40 μg; and lanes 5 and 10, 60 μg. Total binding reaction mixtures were then electrophoresed on nondenaturing 6% polyacrylamide gels. Gels were dried, and overlaid X-ray film was exposed for approximately 2 or 24 h before development. Results presented are from film exposed for 2 h, which showed the same number of bands as film exposed for at least 24 h (data not shown).

FIG. 2.

Binding of PqsR to the pqsA promoter. Radiolabeled DNA containing the pqsA promoter was incubated with E. coli cell lysates containing PqsR. PqsR-containing cell lysate was prepared in the absence (lanes 1 to 5 of panels A, B, and C) or the presence of an ethyl acetate extract of a P. aeruginosa culture (lanes 6 to 10 of panel B) or 20 μM PQS (lanes 6 to 10 of panel C). The total protein concentration of each lysate was determined, and the following amounts of protein were added per reaction: lanes 1 and 6, 0 μg; lanes 2 and 7, 10 μg; lanes 3 and 8, 20 μg; lanes 4 and 9, 40 μg; and lanes 5 and 10, 60 μg. Total binding reaction mixtures were then electrophoresed on nondenaturing 6% polyacrylamide gels. Gels were dried, and overlaid X-ray film was exposed for approximately 3 (B and C) or 72 (A) h.

FIG. 3.

Mapping the pqsR start of transcription. (A) Primer extension analysis of the pqsR transcript. Sequencing reaction mixtures are labeled according to nucleotide (A, C, G, or T), and lane P contains the mixture for the primer extension reaction performed with RNA isolated from P. aeruginosa strain PAO1(pMTP58). Extension products are labeled TS1 and TS2. (B) Repeat of primer extension analysis as described for panel A. Panel B is presented to show that TS1 and TS2 are at the same locations as in panel A. (C) Promoter region of pqsR. The pqsR transcriptional start sites are indicated by bent arrows at TS1 and TS2, and the pqsR ATG start codon is underlined. The −35 and −10 regions of a potential σ⁷⁰-type promoter upstream from TS1 are boxed and labeled. A putative quorum-sensing operator sequence (47) is also boxed, with highly conserved nucleotides in bold.

FIG. 4.

Transcriptional regulation of pqsR in P. aeruginosa. P. aeruginosa strains PAO1 (wild type), PAO-R1 (lasR), PDO111 (rhlR), and MP551 (pqsR) containing plasmid pMWC1003 (pqsR′-lacZ) were cultured as described in Materials and Methods and assayed for β-Gal activity. Data are presented in Miller units as the means ± σⁿ⁻¹ of results from duplicate assays from three separate experiments.

FIG. 5.

LasR and 3-oxo-C₁₂-HSL induce pqsR expression in E. coli. Subcultures were grown for 90 min in the presence or absence of 3-oxo-C₁₂-HSL, and duplicate β-Gal activity assays were performed. Data are presented in Miller units as the means ± σⁿ⁻¹ of results from three separate experiments. Lane 1, E. coli strain DH5α(pMWC1003, pPCS11) without 3-oxo-C₁₂-HSL; lane 2, E. coli strain DH5α(pMWC1003, pPCS11) with 3-oxo-C₁₂-HSL; lane 3, E. coli strain DH5α(pMWC1003, pACYC184) (control).

FIG. 6.

PqsR complements PQS production in a lasR mutant. Ethyl acetate extracts of the indicated cultures were separated by TLC and visualized under UV light. Lanes 1 and 5, synthetic PQS; lane 2, extract from strain PAO1; lane 3, extract from strain PAO-R1(pEX1.8); lane 4, extract from strain PAO-R1(pDSW8).

FIG. 7.

Model of the P. aeruginosa cell-to-cell signaling hierarchy. The details of this model are deliberated in Discussion. Plus and minus symbols indicate positive and negative effects, respectively. The LasI and RhlI signal synthase proteins were left out due to space limitations. Virulence factors include the many virulence determinants and other cell factors that are regulated by the transcriptional activator-signal complexes.