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

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 with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-␤-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.Quorum sensing (QS) has been an emerging research focus in health and environmental sciences during the past decade (2, 5, 9, 37). QS is the population-dependent ability of bacteria to communicate and regulate gene expression through the production, release, and concentration-dependent sensing of signal molecules called autoinducers (9,12,38). A wide range of bacterial processes are now known to be influenced by QS and include bioluminescence, cell density control, toxin production, cell differentiation, exopolysaccharide production, motility, biofilm formation, and virulence factor production (20, 40).Autoinducers are released by cells, diffuse through the extracellular environment, and are "detected" by neighboring cells, often resulting in concentration-dependent changes in gene expression. A major class of autoinducers is the N-acyl homoserine lactones (AHLs) (20). To date, many qualitative and quantitative approaches have been developed to detect AHLs. These include whole-cell-based bioassays using AHL-specific biosensors, thinlayer chromatography, gas chromatography-mass spectrometry (MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), isotopic labeling, and absorbance-based assays (1,6,14,19,21,26,27,34,36,39,41,42,43).A very useful and often applied approach for QS screening is the whole-cell bioassay, which utilizes specific bacterial biosensors (31). It is relatively sensitive and does not require extensive research instrumentation, such as HPLCs and LC-MS.The ␤-galactosidase e...

Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

“…The result demonstrates that several AHLs (C6 -AHL, oxo -C6 -AHL, C8-AHL, C10-AHL, and C12-AHL) were produced by Desulfovibrio vulgaris and other Desulfovibrio species [71,72]. Diverse AHLs were also detected from microbial mats containing a high abundance of SRBs [12].…”

Synergistic effect of quorum sensing genes in biofilm development and PAHs degradation by a marine bacterium

“…19 The AHL production was further confirmed by comparing P. aeruginosa N6P6 crude AHL extract with standard AHLs retention time through HPLC analysis. The genetic basis of AHL production in P. aeruginosa N6P6 was confirmed by amplifying the quorum sensing (QS) genes (lasI and rhlI) coding for AHL synthase with the aid of gene-specific primers.…”

Characterization of Quorum-Quenching Bacteria Isolated from Biofouled Membrane Used in Reverse Osmosis Process

“…CV026 which was used for C4 to C8 AHL detection develops purple pigmentation when exposed to exogenous AHLs with acyl chains shorter than C10 except C4-3-oxo-BHL (McClean et al, 1997;Steindler and Venturi, 2007). NTL4 was used for the detection of AHLs with acyl chains longer than C10 as it can respond to 3-unsubstituted and substituted AHLs with exception of C4-AHL (Cha et al, 1998;Farrand et al, 2002; Zhu et al, 2003;Kawaguchi et al, 2008).…”

“…CV026 which was used for C4 to C8 AHL detection develops purple pigmentation when exposed to exogenous AHLs with acyl chains shorter than C10 except C4-3-oxo-BHL (McClean et al, 1997;Steindler and Venturi, 2007). NTL4 was used for the detection of AHLs with acyl chains longer than C10 as it can respond to 3-unsubstituted and substituted AHLs with exception of C4-AHL (Cha et al, 1998;Farrand et al, 2002; Zhu et al, 2003;Kawaguchi et al, 2008).Among a total of 505 bacteria which were isolated from fouled RO membranes collected from a full-scale water treatment plant (Veolia Water Solutions & Technologies Korea), Daesan, Chungbuk, Korea , 34 strains presented QQ activity in parallel streaking assay. Based on the disc-diffusion assay, 6 isolates were chosen to have high QQ activity against BHL (C4), HHL (C6), DHL (C10), and OdDHL (3-oxo-C12) (Fig.…”