QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa

Abstract

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR-LasI and RhlR-RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Figure 1

Relationship of the qscR gene and gene product to other genes and their products. (Upper) Alignment of the QscR sequence with SolR, RhlR, LasR, and LuxR. Conserved amino acids are shaded in black. Gray shading indicates that 100% of the residues are similar at that position. The alignment was constructed by using the clustal w multiple alignment program and the degree of residue shading was determined by using Boxshade at 1.0 setting (37). The sequences used in the alignment are R. solanacearum SolR (GenBank accession no. AF021840), P. aeruginosa RhlR (L08962), P. aeruginosa LasR (M59425), and V. fischeri LuxR (M96844). (Lower) A map of qscR and the genes flanking qscR in the P. aeruginosa genome. In the P. aeruginosa genome annotation, genes designated A2–G2 are called phzA2-G2 based on similarity to genes in the phz operon (http://www.pseudomonas.com). The genes in the phzA2-G2 operon are each over 98% identical at the DNA sequence level to the bona fide phzA-G genes. Other phenazine-producing species of Pseudomonas have operons with related genes. The qscR gene is called phzR in the P. aeruginosa genome annotation.

Figure 2

The influence of qscR on quorum-controlled factors in P. aeruginosa. (A) Pyocyanin levels in P. aeruginosa PAO1 (□), PAOR3 (▴), and PAOR3 with the qscR plasmid pKL9 (○). β-Galactosidase in P. aeruginosa PAO1 (□), and PAOR3 (▴) containing the phzABC-lacZ plasmid, pMW303 (B), the hcnAB-lacZ plasmid, pMW301 (C), and the qsc105-lacZ plasmid, pMW105B (D).

Figure 3

Expression of the lasR–lacZ (pSC10) (A), rhlR–lacZ (pMW304) (B), lasI–lacZ (pSC11) (C), and the rhlI–lacZ (pMW305) (D) transcriptional fusions in P. aeruginosa PAO1 (□), PAO1 with exogenously added 2 μM 3OC₁₂-HSL (○), and PAOR3 (▴).

Figure 4

The concentrations of 3OC₁₂-HSL (A) and C₄-HSL (B) in cultures of P. aeruginosa PAO1 (□) and PAOR3 (▴).

Figure 5

Virulence of the P. aeruginosa qscR mutant in fruit flies. Death of flies over time when fed P. aeruginosa PAO1 (□), PAOR3 (▴), or E. coli CM830 (○). The values are the averages from 10 replicate experiments, each with 9–12 flies, and the SEM is shown for each value plotted. The parent and mutant bacteria survived equally well in the dry culture vials during the course of the experiments (data not shown). The number of bacteria recovered from flies just after death was 12 (± an SEM of 2) × 10⁶ PAO1, and 8 (±3) × 10⁶ PAOR3.