Quorum sensing (QS) has been an emerging research focus in health and environmental sciences during the past decade (2
). 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
). 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
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, thin-layer chromatography, gas chromatography-mass spectrometry (MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), isotopic labeling, and absorbance-based assays (1
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 β-galactosidase expression system has been used as a specific indicator of gene expression (17
). The bacterial reporter strain Agrobacterium tumefaciens
NTL4(pCF218)(pCF372) contains the β-galactosidase gene driven by a traI
promoter, allowing the expression of β-galactosidase to be regulated by the presence of QS signals (AHLs). In the presence of the substrate 5-bromo-4-chloro-3-indolyl-β-d
-galactopyranoside (X-Gal), β-galactosidase enzymatically cleaves X-Gal, which results in its conversion to a blue precipitate when active forms of AHLs are present. Accumulation of the blue precipitate is then detectable by spectral absorbance at 635 nm.
A rapidly developing area in the study of QS involves the detection of autoinducer activities from bacterial communities under natural conditions. Whole-cell bioassays are often used for screening gram-negative bacterial colonies that produce AHLs. However, whole-cell bioassays are constrained by several limitations: (i) the requirement of a time-consuming cell conditioning step prior to the start of the bioassay, (ii) the lengthy incubation times (i.e., at least 24 h for detection of AHLs), and (iii) the sensitive adjustments in cell densities needed (e.g., of each well of a 96-well plate or each test tube) to calculate relative activities of luminescence or absorbance. Therefore, a simple and rapid assay that is both sensitive and relatively robust (under environmental conditions) is needed for the detection of AHLs extracted from natural systems.
In the present approach, we have reduced these limitations by developing a cell-free assay system to detect AHL QS signals. Cell-free lysates were derived from the reporter bacterium A. tumefaciens
NTL4(pCF218)(pCF372) and, without any addition, contained all the necessary cellular components for in vitro gene expression and translation (e.g., 70S ribosome; tRNAs; aminoacyl-tRNA synthetases; initiation, elongation, and termination factors; amino acids; ATP; GTP; and cofactors such as Mg2+
). The β-galactosidase expression system was shown to increase in stoichiometric proportion to the concentration of AHLs. Using this system, many samples could be rapidly screened for the presence of AHLs by simple addition of a cell-free lysate and reporter substrate. AHLs were then detected within 3 h using a microplate reader, spectrophotometer, or luminometer.