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Applied and Environmental Microbiology, June 2008, p. 3667-3671, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02869-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Screening of Quorum-Sensing Signal N-Acyl Homoserine Lactones by an In Vitro Cell-Free Assay{triangledown}

Tomohiro Kawaguchi,* Yung Pin Chen, R. Sean Norman, and Alan W. Decho

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

Received 19 December 2007/ Accepted 11 April 2008

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 β-galactosidase upon addition of exogenous AHLs. We have shown that β-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-β-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°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.

* Corresponding author. Mailing address: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208. Phone: (803) 777-4347. Fax: (803) 777-3391. E-mail: kawaguch{at}gwm.sc.edu

{triangledown} Published ahead of print on 18 April 2008.

Applied and Environmental Microbiology, June 2008, p. 3667-3671, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02869-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yang, Y.-H., Kim, T.-W., Park, S.-H., Lee, K., Park, H.-Y., Song, E., Joo, H.-S., Kim, Y.-G., Hahn, J.-S., Kim, B.-G. (2009). Cell-Free Escherichia coli-Based System To Screen for Quorum-Sensing Molecules Interacting with Quorum Receptor Proteins of Streptomyces coelicolor. Appl. Environ. Microbiol. 75: 6367-6372 [Abstract] [Full Text]  


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