Regulation of <em>Pseudomonas</em> Quinolone Signal Synthesis in <em>Pseudomonas aeruginosa</em>

Dana Wade

M Calfee

Edson Rocha

Elizabeth Ling

Elana Engstrom

James Coleman

Everett Pesci

Department of Microbiology and Immunology, The Brody School of Medicine at East Carolina University, 600 Moye Blvd., Greenville, North Carolina 27834

^{Corresponding author. Mailing address: Department of Microbiology and Immunology, East Carolina University School of Medicine, BT 132, 600 Moye Blvd., Greenville, NC 27834. Phone: (252) 744-2351. Fax: (252) 744-3535. E-mail:}ude.uce.liam@eicsep.

Received 2005 Feb 1; Accepted 2005 Mar 29.

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 β-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.

Abstract

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium that can infect insects, plants, and animals. As an opportunistic pathogen of humans, P. aeruginosa causes acute infections in immunocompromised individuals and chronic lung infections in cystic fibrosis patients. Such infections are made possible through the production of an arsenal of virulence factors, many of which are regulated by cell-to-cell signals (see reference 36 for a review). P. aeruginosa produces at least three small compounds that function as intercellular communication signals. The acyl homoserine lactone signals, N-(3-oxododecanoyl) homoserine lactone (3-oxo-C₁₂-HSL) and N-butyryl homoserine lactone (C₄-HSL), have been well studied and function in combination with the LuxR homologs LasR and RhlR, respectively (16, 30, 31, 33, 34). Together, these quorum-sensing signals control 6 to 11% of the P. aeruginosa genome (44, 46, 48). The third P. aeruginosa intercellular signal is a quinolone compound that was identified as 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal [PQS]) (37). This signal controls multiple virulence factors and is intertwined in the quorum-sensing cascade, where it appears to be a regulatory link between the las and rhl quorum-sensing systems (14, 27). PQS is produced in the lungs of cystic fibrosis patients infected with P. aeruginosa (9) and is required for virulence in nematodes, plants, and mice (5, 15, 23, 25, 40). PQS also induces apoptosis and decreases viability in eukaryotic cells (4).

The synthesis of PQS requires several putative enzymes encoded by the pqsABCDE, phnAB, and pqsH operons (10, 15). In addition, PqsR (also known as MvfR [5]) is a LysR-type regulator required for the synthesis of PQS (10, 15) and at least 55 related 4-quinolone compounds (24). A recent genomics study indicated that 143 genes were differentially regulated in a pqsR mutant (12), demonstrating the global regulatory nature of the PQS system. Initial studies on the expression of the PQS synthetic genes indicated that the genes are governed by a complex regulatory scheme. PqsR was found to have a positiveeffect on the transcription of the pqsABCDE and phnAB operons (5, 26). In addition, the transcription of the pqsABCDE promoter was positively regulated by LasR-3-oxo-C₁₂-HSL and negatively regulated by RhlR-C₄-HSL (26). Whether the LasR, RhlR, and PqsR transcriptional regulators were acting in a direct or indirect manner to control pqsABCDE expression was not determined. In this study, the interactions that occur at the pqsABCDE promoter were investigated. We demonstrate that the effects of LasR and RhlR on the pqsABCDE operon occur indirectly through PqsR. We also show that PqsR, but not LasR or RhlR, binds directly to the pqsABCDE promoter and that this binding is augmented by the presence of PQS.

Acknowledgments

This work was supported by a research grant from the National Institute of Allergy and Infectious Diseases (grant R01-AI46682).

We thank J. Farrow and C. Pesci for help in manuscript preparation and thoughtful insight.

Acknowledgments

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