QUORUM SENSING IN VIBRIO-ANGUILLARUM - CHARACTERIZATION OF THE VANI VANR LOCUS AND IDENTIFICATION OF THE AUTOINDUCER N-(3-OXODECANOYL)-L-HOMOSERINE LACTONE/

Certain gram-negative pathogens are known to control virulence gene ex pression through cell-cell communication via small diffusible signal m olecules termed autoinducers, This intercellular signal transduction m echanism termed quorum sensing depends on the interaction of an N-acyl homoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators, Vibrio anguillarum is a gram-negative pathogen capable of causing a te rminal hemorrhagic septicemia known as vibriosis in fish such as rainb ow trout. in this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguilla rum culture supernatants stimulated bioluminescence in a recombinant l ux-based Escherichia coli AHL biosensor strain, whereas they both stim ulated and inhibited AHL-mediated violacein pigment production in Chro mobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid c hromatography and high-resolution tandem mass spectrometry we identifi ed the major V, anguillarum, AHL as N-(3-oxodecanoyl)-L-homoserine lac tone (ODHL), a structure,which was unequivocally confirmed by chemical synthesis, Tile gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AWL synthases. Further sequencing downstream of vanI revealed a second gen e (vanR) related to the LuxR family of transcriptional activators, Alt hough deletion of vanI abolished ODHL synthesis, no reduction of eithe r metalloprotease production or virulence in a fish infection model wa s observed. However, the vanI mutant remained capable of weakly activa ting both bioluminescence and violacein in the E. call and C, violaceu m biosensors, respectively, indicating the existence of additional lay ers of AHL-mediated regulatory complexity.