Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules.
2-Alkyl-4-quinolones (AHQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) are quorum sensing signal molecules. Here, we describe methods for AHQ detection, tentative identification and quantification, which employ a lux-based Pseudomonas aeruginosa AHQ biosensor strain. The protocol describes both thin-layer chromatography (TLC) and microtiter plate assays, which use bioluminescence or the green color of pyocyanin as detection end points. Organic solvent extracts of bacterial cells or cell-free culture supernatants are chromatographed on TLC plates, which are dried and overlaid with the AHQ biosensor. AHQs appear as both luminescent and green spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AHQ biosensor. Light output is proportional to the AHQ content of the sample. The assays described take approximately 2 days to complete, are simple to perform, do not require sophisticated instrumentation and are highly amenable to screening large numbers of bacterial samples. However, apart from PQS and HHQ in P. aeruginosa, definitive AHQ identification will require additional MS and NMR analyses.
Fletcher
Matthew P
MP
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK.
Diggle
Stephen P
SP
Cámara
Miguel
M
Williams
Paul
P
D015374:Biosensing Techniques / Q000379:methods*; D002855:Chromatography, Thin Layer; D008163:Luminescent Measurements / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D015363:Quinolones / Q000302:isolation & purification*; D015398:Signal Transduction
C407944:2-heptyl-3-hydroxy-4-quinolone; D015363:Quinolones