Detection of N-acyl homoserine lactones using a traI-luxCDABE-based biosensor as a high-throughput screening tool

Abstract

Background: Bacteria use N-acyl homoserine lactone (AHL) molecules to regulate the expression of genes in a density-dependent manner. Several biosensors have been developed and engineered to detect the presence of all types of AHLs.

Results: In this study, we describe the usefulness of a traI-luxCDABE-based biosensor to quickly detect AHLs from previously characterized mutants of Burkholderia cenocepacia and Pseudomonas aeruginosa in both liquid and soft-agar co-culture assays in a high-throughput manner. The technique uses a co-culture system where the strain producing the AHLs is grown simultaneously with the reporter strain. Use of this assay in liquid co-culture allows the measurement of AHL activity in real time over growth. We tested this assay with Burkholderia cenocepacia and Pseudomonas aeruginosa but it should be applicable to a broad range of gram negative species that produce AHLs.

Conclusion: The co-culture assays described enable the detection of AHL production in both P. aeruginosa and B. cenocepacia and should be applicable to AHL analysis in other bacterial species. The high-throughput adaptation of the liquid co-culture assay could facilitate the screening of large libraries for the identification of mutants or compounds that block the synthesis or activity of AHLs.

Figure 1

Detection of AHL using the A. tumefaciens A136 (pCF218) (pMV26) as biosensor in liquid co-culture assay. Detection and temporal expression of AHL in liquid co-culture assay for B. cenocepacia (A) and P. aeruginosa strains (B). Values shown are the mean ± standard deviation of at least four replicates.

Figure 2

Detection of AHLs using the A. tumefaciens A136 (pCF218) (pMV26) as biosensor on soft agar co-culture assay. Detection of AHLs in a soft agar co-culture assay for B. cenocepacia (A) and P. aeruginosa strains (B). Panel A: 1, K56-2cepI cciIb; 2, K56-2cciI; 3, K56-2cepR cciIR; 4, K56-2cciIR; 5, K56-dI2; 6, K56-2cciR; 7, K56-I2; 8, K56-R2; 9, K56-2. Panel B: 1, PAO1rhlI lasI; 2, PAO1rhlR; 3, PAO1lasI; 4, PAO1rhlI; 5, PAO1lasR; 6, PAO1.

Figure 3

Comparison of the detection of AHLs using the A. tumefaciens A136 (pCF218) (pMV26) as biosensor with different microtiter plate formats. Temporal expression of AHL in liquid co-culture assay by B. cenocepacia strains in 96- (A) and 384-well formats (B). Values shown are the mean ± standard deviation of at least 3 replicates.