Project

  Introduction

Cell signaling and quorum sensing in P. aeruginosa is mediated by the autoinducers PAI-1 and PAI-2, so these molecules are excellent indicators that this bacterium is present. ^[1]. The production of PAI-1 and PAI-2 are specific to the las and the rhl systems respectively^[2]. Transcriptional activation of target genes can be amplified up to 1000-fold due to the binding of the R-protein autoinducer/dimer complex to its respective target promoter^[3]. Therefore, our detection system uses the specificity of these two autoinducer-response systems as a definitive means of detecting P. aeruginosa.

Figure 1: Activation of the induced promoters of P. aeruginosa Las and Rhl sequences by autoinducer/R-protein complexes (transcriptional regulation)

Our detection system incorporates LasR and RhlR receptors (R-proteins) for the autoinducers PAI-1 and PAI-2. As previously reported, LasR/PAI-1 and RhlR/PAI-2 protein complexes act primarily as transcriptional factors^[4]. Therefore, our platform will utilize inducible promoters from the genome of P. aeruginosa as indicated in Figure 2. These promoters are induced by their respective ligand-bound dimeric R-protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. In our proof-of-concept system, the inducible promoters drive the expression of reporter proteins, such as GFP or RFP, to report the presence of P. aeruginosa autoinducer molecules through a readout that is easily quantified. Future implications of this concept could include reporter genes that are detectable by other means, such as a simple visual color-change system.

Figure 2: Coupling the induced promoters of P. aeruginosa with reporting constructs (inserts 1 and 2)

In our system, the lasR and rhlR genes are expressed from a high efficiency constitutive promoter and ribosome binding site (RBS). Given this high steady-state expression of LasR and RhlR proteins, we predict that the autoinducers PAI-1 and PAI-2 will be rate-limiting for the induction of the reporter genes. Therefore, reporter gene expression should commence rapidly upon the binding of PAI-1 or PAI-2 to their respective R-protein.