PMC

Simplified schematic representation of the AQ-dependent QS in P. aeruginosa (modified from ). HHQ, the immediate precursor of PQS, drives the expression of the pqsABCDE operon via PqsR(MvfR) and is also converted to PQS by the action of the monooxygenase, PqsH. PQS also drives pqsABCDE expression via PqsR. PqsE positively regulates biofilm, swarming virulence and secondary metabolite gene expression but negatively regulates pqsABCDE expression. PQS also binds ferric iron which results in the induction of high affinity siderophore iron transport genes. AHL and AQ-dependent QS are linked because LasR/3-oxo-C12-HSL is required for maximal expression of pqsH and pqsR whereas pqsR and pqsABCDE are repressed by RhlR/C4-HSL.

A. Schematic representation of the pqs locus in P. aeruginosa PAO1 wild type and the IPTG-inducible pqsE strain, pqsEind. The Ω interposon (Sm^R/Spc^R) is from plasmid pHP45Ω: the lacI^Q repressor is derived, together with the Ptac promoter, from plasmid pME6032. B. Activity of the PpqsA::lux promoter fusion. The activity of the PpqsA promoter was monitored during growth in PAO1 wild type, pqsEind, rhlR and pqsEind rhlR double mutants. The maximal expression levels reached during the late exponential phase of growth are shown. Where indicated (+), 1 mM IPTG was added to the growth medium. Error bars are calculated from three independent experiments. C. Concentration of HHQ (grey bars) and PQS (white bars) determined by LC-mass spectrometry in PAO1 wild type and the pqsEind mutant. The AQs were extracted from overnight cultures grown in LB broth; where indicated (+), 1 mM IPTG was added to the growth medium. The average of the results from three independent experiments is shown and error bars represent two standard deviations from the mean.

A. Pyocyanin produced by PAO1 and both pqsEind and pqsA pqsEind mutants. Bacterial cultures were grown in LB broth (grey bars) or LB broth supplemented with 1 mM IPTG (white bars), and pyocyanin was extracted after 16 h growth (early stationary phase). B. Western blot analysis of Lectin A in cell extracts of PAO1 and both pqsEind and pqsA pqsEind mutants. Proteins were extracted from cultures grown for 16 h in LB broth to an OD₆₀₀ of 2.5 (early stationary phase of growth), with (+) or without (−) 1 mM IPTG. An extract from P. aeruginosa PAO1 lecA mutant (lecA::lux) was used as a negative control. C. Swarming assays performed with PAO1 and both pqsEind and pqsA pqsEind mutants in the presence or absence of 1 fmM IPTG. D. Biofilm formation on stainless steel coupons by PAO1 and both pqsE and pqsA pqsEind mutants. A representative picture of the biofilm formed by each strain is also shown. For A and D, the average of the results from three independent experiments is reported with standard deviations.

PqsE restores virulence in nematode, plant and animal infection models in the absence of AQs. A. Caenorhabditis elegans killing assay showing the percentage of nematode survival after 1–6 days of exposure to the P. aeruginosa PAO1 wild type, pqsA pqsEind mutant and pqsA pqsEind mutant transformed with the vector control, pUCP18 or pUCPpqsE respectively. The average of four independent experiments is reported with standard deviation. B. Virulence of the wild type, pqsA mutant and pqsA mutant complemented with pqsE in the lettuce leaf virulence assay. The number of bacterial cells (as colony forming units, cfu) present in 1 mg of lettuce midrib 5 days post injection is shown. Error bars were calculated from five independent experiments. A representative picture of infected midribs is also shown for each strain. C and D. Mouse acute burn wound infection showing the survival rate over time (days after burn/infection) for mice infected with (C) the P. aeruginosa wild type (▵), pqsA (□) and pqsE (○) mutants; 15 mice per mutant and (D) the P. aeruginosa pqsA mutant (□) and the pqsA mutant transformed with either pUCP18 (○) or pUCPpqsE (▵); nine mice per mutant.