- Automated luminometer-spectrometer plate reader.
- UV/Vis spectrophotometer.
- Plastic microcuvettes for UV/Vis spectrophotometer.
3 Methods
3.1 Rationale of the
Primary Screening
The screening system for inhibitors ofP. aeruginosaQS is based on
a biosensor strain (PA14-R3) able to detect the QS signal molecule
3OC 12 -HSL [16]. The PA14-R3 biosensor is available from the
authors upon request.
The PA14-R3 strain carries a nonfunctional allele of thelasI
gene, and is thus unable to synthesize 3OC 12 -HSL; however, it can
respond to exogenous 3OC 12 -HSL provided either through supply
of the purified molecule or by cocultivation with a wild typeP.
aeruginosaproficient in 3OC 12 -HSL production, such as the PA14
strain. The screening system is detailed in Fig.1. The 3OC 12 -HSL
signal synthesized by the wild type PA14 diffuses into the PA14-R3
biosensor and induces bioluminescence emission. The addition of a
molecule with inhibitory activity towards any process related to the
3OC 12 -HSL-dependent QS system, namely, 3OC 12 -HSL synthe-
sis, transport, and perception, will reduce light emission by the
biosensor with respect to a control coculture grown in the absence
of any chemical compound.Fig. 1Schematic representation of the PA14/PA14-R3 cocultivation screening system. The wild type PA14
produces 3OC 12 -HSL signal molecules that induce bioluminescence emission in the biosensor strain PA14-R3.
PA14-R3 is a PA14 derivative in which a transcriptional fusion between the LasR-dependentrsaLpromoter
(PrsaL) and theluxCDABEoperon was integrated at theattBneutral site of the chromosome. In addition, in
order to avoid self-activation of the reporter strain, thelasIgene, encoding the 3OC 12 -HSL synthase LasI, was
inactivated by transposon insertion (Tn). Molecules interfering with different steps of thelasQS system are
expected to cause a reduction in bioluminescence in comparison to the untreated control. Modified from [16]
290 Giordano Rampioni et al.