Structure-activity analysis of the Pseudomonas quinolone signal molecule

Abstract

We synthesized a range of PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone) analogues and tested them for their ability to stimulate MvfR-dependent pqsA transcription, MvfR-independent pyoverdine production, and membrane vesicle production. The structure-activity profile of the PQS analogues was different for each of these phenotypes. Certain inactive PQS analogues were also found to strongly synergize PQS-dependent pyoverdine production.

FIG. 1.

Structures of the PQS analogues used in this work.

FIG. 2.

Stimulation of MvfR-dependent pqsA promoter activity by the PQS analogues. Cultures of E. coli strain DH5α containing pEAL08-2 were grown in LB with good aeration at 37°C using shake flasks. Each culture contained 60 nM PQS analogue, and the β-galactosidase activity (corrected for the culture optical density at 600 nm [OD₆₀₀]) was measured after 2.5 h, essentially as described by Cugini et al. (4). No effect on growth was observed for any of the analogues. DMSO (−) was added as a control. The data represent the averages and standard deviations from the results of 3 independent biological repeats.

FIG. 3.

Stimulation of pyoverdine production by the PQS analogues. An overnight culture of P. aeruginosa strain DH125 was used to inoculate LB to a starting OD₆₀₀ of 0.05. The PQS analogues (or, as a control, DMSO) were added to a final concentration of 30 μM (white bars), 60 μM (light gray bars), or 90 μM (dark gray bars), and the samples were grown for 8 h with good aeration at 37°C. After this, the OD₆₀₀ was recorded, and the samples were clarified by centrifugation (10,000 × g, 10 min, 20°C). Pyoverdine in the supernatant was quantified as previously described (8) at 405 nm. The data represent the averages and standard deviations from the results of 3 independent biological repeats. None of the analogues affected growth at any of the concentrations tested. P, PQS; −, DMSO control.

FIG. 4.

Effect of iron availability on AHQ-dependent pyoverdine production. Cultures were grown, and pyoverdine was assayed as described in the legend to Fig. 3, except that the indicated AHQs were present at a 90 μM concentration and the medium used was LB supplemented with 300 μM FeCl₃ (A), LB alone (B), or deferrated LB (C). ND, not determined. The data represent the averages and standard deviations from the results of 3 independent biological repeats.

FIG. 5.

Nonactive PQS analogues synergize PQS-dependent pyoverdine stimulation. Cultures of P. aeruginosa strain DH125 were incubated for 8 h in the absence of PQS (−), with 25 μM PQS (PQS), or with 25 μM PQS and 75 μM indicated analogues. Pyoverdine was then assayed in the culture supernatant at 405 nm (8). The data represent the averages and standard deviations from the results of 3 independent biological repeats.

FIG. 6.

Membrane vesicle formation in the presence of PQS analogues. Cultures of PA14 or the isogenic pqsB mutant (DH125) were grown with good aeration (200 rpm in 250-ml flasks) at 37°C in 20 ml brain heart infusion (BHI) medium. The DH125 cultures contained DMSO alone (−) or 50 μM indicated analogues. After 18 h, the cultures were harvested and clarified by sedimentation at 5,000 × g (20°C) for 10 min, and the clarified supernatants were passed through a 0.2-μm filter. Aliquots (1-ml volume) of the filtered supernatant were sedimented for 2.5 h (4°C) at 800,000 × g in a Beckman Optima benchtop ultracentrifuge (MLA-130 rotor). The supernatant was removed by aspiration, and the pellet was washed once in 50 mM NaCl. The pellets were air dried and resuspended in 30 μl water. The phospholipid content of each pellet was measured using the ammonium ferrothiocyanate method of Stewart (20). The data represent the averages from the results of 2 independent biological repeats. WT, wild type (the MV content of PA14); P, PQS.

FIG. 7.

Quantitative comparison of PQS analogue activity in MvfR-dependent and MvfR-independent assays. The induction ratios (activity in the presence of the analogue/activity in the absence of the analogue) were calculated using the data shown in Fig. 2 and 3 and then plotted against one another. The identities of the active analogues in cluster I are indicated.