Response of PqsR^CBD ligand binding site mutants to AQs.

<p>(<b>A</b>) PqsR-6His is functional in <i>P. aeruginosa</i>. The <i>pqsR</i> gene with or without a 6His tag on pME6032 was introduced into a <i>P. aeruginosa pqsR</i> deletion mutant (Δ<i>pqsR</i>) containing a chromosomal miniCTX::<i>pqsA'-lux</i> reporter gene fusion. Relative PqsR activity was determined as a % of the maximum bioluminescence produced by the miniCTX::<i>pqsA'-lux</i> reporter fusion in a wild type <i>P. aeruginosa</i> PAO1 background carrying the empty pME6032 vector; (<b>B</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with one of each of the 13 site-specific mutations introduced into the PqsR^CBD ligand-binding pocket. Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to PAO1 Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> expressing the PqsR-6His protein (WT); (<b>C</b>) Western blot analysis confirming expression of each of the PqsR-6His mutant proteins; (<b>D</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with either the gene coding for PqsR-6His or one of the 13 site-specific mutants and supplemented with either HHQ or PQS (40 µM). Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain expressing PqsR-6His.