| Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay. | |
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MedLine Citation:
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PMID: 18424536 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Tomohiro Kawaguchi; Yung Pin Chen; R Sean Norman; Alan W Decho |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, U.S. Gov't, Non-P.H.S. Date: 2008-04-18 |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 74 ISSN: 1098-5336 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2008 Jun |
Date Detail:
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Created Date: 2008-06-16 Completed Date: 2008-07-23 Revised Date: 2010-09-22 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 3667-71 Citation Subset: IM |
Affiliation:
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Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, USA. kawaguch@gwm.sc.edu |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Acyl-Butyrolactones
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analysis* Biosensing Techniques / methods* Cell-Free System Densitometry Galactosides / metabolism Indoles / metabolism Luminescent Agents / metabolism Luminescent Measurements Quorum Sensing Rhizobium radiobacter / enzymology Sensitivity and Specificity Time Factors beta-Galactosidase / metabolism |
| Chemical | |
Reg. No./Substance:
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0/Acyl-Butyrolactones; 0/Galactosides; 0/Indoles; 0/Luminescent Agents; 7240-90-6/5-bromo-4-chloro-3-indolyl beta-galactoside; EC 3.2.1.23/beta-Galactosidase |
| Comments/Corrections | |
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