Document Detail


Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay.
MedLine Citation:
PMID:  18424536     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Tomohiro Kawaguchi; Yung Pin Chen; R Sean Norman; Alan W Decho
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-04-18
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  74     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-06-16     Completed Date:  2008-07-23     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3667-71     Citation Subset:  IM    
Affiliation:
Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, USA. kawaguch@gwm.sc.edu
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MeSH Terms
Descriptor/Qualifier:
Acyl-Butyrolactones / analysis*
Agrobacterium tumefaciens / enzymology
Biosensing Techniques / methods*
Cell-Free System
Densitometry
Galactosides / metabolism
Indoles / metabolism
Luminescent Agents / metabolism
Luminescent Measurements
Quorum Sensing
Sensitivity and Specificity
Time Factors
beta-Galactosidase / metabolism
Chemical
Reg. No./Substance:
0/Acyl-Butyrolactones; 0/Galactosides; 0/Indoles; 0/Luminescent Agents; EC 3.2.1.23/beta-Galactosidase; V595OG374W/5-bromo-4-chloro-3-indolyl beta-galactoside
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