Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay.
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.
Kawaguchi
Tomohiro
T
Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, USA. kawaguch@gwm.sc.edu
Chen
Yung Pin
YP
Norman
R Sean
RS
Decho
Alan W
AW
D054742:Acyl-Butyrolactones / Q000032:analysis*; D016960:Agrobacterium tumefaciens / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods*; D002474:Cell-Free System; D003720:Densitometry; D005697:Galactosides / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D049408:Luminescent Agents / Q000378:metabolism; D008163:Luminescent Measurements; D053038:Quorum Sensing; D012680:Sensitivity and Specificity; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D054742:Acyl-Butyrolactones; D005697:Galactosides; D007211:Indoles; D049408:Luminescent Agents; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside