FIG 2.

LasR does not affect the transcription of nadA. (A) Primer extension analysis of the nadA transcript (lane Ext). Lanes labeled A, C, G, and T contain DNA sequencing ladders that were generated by cycle sequencing and that terminate at the corresponding nucleotide positions. Figure 1A shows the relative location of the primer extension product identified by the arrowhead. (B) Radiolabeled DNA fragments corresponding to pqsR promoter fragment pqsR-P2 or pqsR-P2 with a mutated LasR binding site were incubated in the presence or absence of purified LasR and 5 μM 3-oxo-C₁₂-HSL. Samples of binding reaction products were analyzed on nondenaturing acrylamide gels, and the position of DNA fragments was visualized by autoradiography. (C) P. aeruginosa strain PAO1 carrying either a plasmid containing the wild-type nadA promoter fused to lacZ (open bars) or a plasmid containing an nadA promoter with a mutated LasR binding site fused to lacZ (striped bars) was grown in LB medium for 6 h, and then cultures were assayed for β-Gal activity. (D) P. aeruginosa strain PAO1 carrying either a plasmid containing the wild-type pqsR promoter fused to lacZ (open bars) or a plasmid containing a pqsR promoter with a mutated LasR binding site fused to lacZ (striped bars) was grown in LB medium for 6 h, and then cultures were assayed for β-Gal activity. β-Gal data are presented in Miller units as the means ± standard deviations of results from duplicate assays from at least three separate experiments.