Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay

Abstract

A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of beta-galactosidase upon addition of exogenous AHLs. We have shown that beta-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (-80 degrees C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.

FIG. 1.

Confirmation of β-galactosidase expression in in vitro cell-free solution. (A) Components required for reactions 1 through 3. These reactions include binding of AHLs with the reporter protein TraR (reaction 1), binding of the TraR/AHL complex to the traR promoter driving the expression of β-galactosidase (reaction 2), and translation of mRNA for the synthesis of β-galactosidase (reaction 3). CFL, cell-free lysate. (B) Inhibition of reaction 1 with high temperature. (C) Inhibition of reactions 2 and 3 by streptomycin. Bars indicates means ± standard deviations (n = 3).

FIG. 2.

Dose-response curve of β-galactosidase expression in a cell-free lysate with addition of C₈-AHL. Points represent mean values of three samples. Error bars represent standard deviations.

FIG. 3.

Optimization of cell-free assay conditions. (A) Crude protein concentrations in a cell-free extract solution. (B) Optimum pH for a cell-free assay. (C) Incubation times for cell-free assays. Values (points or bars) indicate means ± standard deviations (n = 3).