A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds.
Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2-dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wild-type Vibrio harveyi but exhibited no response to culture filtrates from V. harveyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent Kd = 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed.
Rajamani
Sathish
S
Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA.
Zhu
Jinge
J
Pei
Dehua
D
Sayre
Richard
R
D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006714:Homoserine / Q000031:analogs & derivatives* / Q000032:analysis; D007783:Lactones / Q000032:analysis*; D015398:Signal Transduction
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D007783:Lactones; C090749:LuxP protein, Vibrio; C091088:N-octanoylhomoserine lactone; D049452:Green Fluorescent Proteins; D006714:Homoserine