A Simple Biosensor-Based Assay for Quantitative Autoinducer-2 Analysis

Abstract

Bacteria produce and react to interspecies signaling molecules in order to control the expression of genes that are particularly beneficial when they are expressed by a bacterial community. In addition to intraspecies communication, the signaling molecule autoinducer-2 (AI-2) can also serve for interspecies communication between Gram-positive and Gram-negative bacteria and is therefore of particular interest. The analysis and quantification of AI-2 are essential for understanding population density-dependent changes in bacterial behavior and pathogenicity. However, currently available bioassays for AI-2 quantification are rather complex, have narrow detection ranges, and are very sensitive to trace components of, for example, growth media. To facilitate and improve the detection of AI-2, we have developed an Escherichia coli biosensor-based assay that is sensitive, cheap, fast, robust, and reliable in the quantification of biologically active AI-2. The bioassay is based on an lsr promoter-fluorescent reporter gene fusion cassette that we chromosomally integrated in a biosensor strain, but the cassette can also be used in a low-copy number plasmid for the application in other Gramnegative bacterial species. We show here that AI-2 quantification was possible in a concentration range from 400 nM to 100 μM and that a critical interpretation of the kinetics of the measurements can reveal sugar interference. With the help of our biosensor strain, coculture experiments were done to test the capability and kinetics of AI-2 secretion by various Gram-negative bacteria in real time. Finally, calibration curves were used to calculate the absolute AI-2 concentration in cell-free bacterial samples.

Anti-Quorum-Sensing Potential of Ethanolic Extracts of Aromatic Plants from the Flora of Cyprus

“…When bacteria reach a critical concentration, they release signal molecules called AIs [ 37 ]. QS is often employed to regulate beneficial genes expressed by a bacterial community, including genes implicated in virulence, biofilm formation, swarming and swimming motilities, stress resistance, and antibiotic resistance [ 39 , 40 ]. Therefore, inhibition of QS communication among bacteria could be used as an alternative strategy to fight multi-drug-resistant bacteria, while any compound that is able to inhibit AI activity without interfering with the growth rate of bacteria can be considered to be a potential QS inhibitor [ 37 ].…”

Anti-quorum Sensing Potential of Ethanolic Extracts of Aromatic Plants From Cyprus Flora

“…In the past years, it has been demonstrated that both Gand G + bacteria use QS to coordinate gene expression in a cell density-dependent manner [37]. When bacteria reach a critical concentration they release signal molecules which are called AIs [36].QS is often employed to regulate genes that are particularly useful when expressed by a bacterial community, including genes that are implicated in virulence, biofilm formation, swarming and swimming motility, stress resistance, and resistance to antibiotics [38,39]. Therefore, inhibition of QS communication between bacteria could be used as an alternative strategy to fight multidrug resistance bacteria while any compound able to inhibit AI activity without growth inhibition can be considered to be a promising QS inhibitor [36].…”

Microbiota-Accessible Boron-Containing Compounds in Complex Regional Pain Syndrome

“…In addition, B has recently been declared an essential element involved in the synthesis of the autoinducer-2 (AI-2) quorum sensing (QS) system. At the same time, active AI-2B is generated by the addition of borate to the AI-2 precursor, being able to amplify the activity of AI-2 and support the secretion of extracellular polymeric substances [122,123].…”