Rapid Screening of Quorum Sensing signal N-acyl homoserine Lactones by an In Vitro Cell-Free Assay

Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC. 29208. USA

	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 -galactosidase upon addition of exogenous AHLs. We have shown that -galactosidase expression is possible in cell-free solution (lysate from Agrobacterium tumefaciens NTL4 (pCF218)(pCF372) culture). Assay detection limits using chromogenic substrate X-Gal ranged from approximately 100 nM to 300 nM depending on the specific AHL. Substitution (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°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 metagenomics libraries.