Bioassay for interference of AHL-dependent gene regulation in Pectobacterium atrosepticum and modulation of LuxI and LuxR by N-acylhomoserine lactone degrading rhizobacteria

Quorum sensing is a communication system that allows bacteria to monitor their population density through the production and sensing of signal molecules. In Gram-negative bacteria, signal molecules include acyl-homoserine lactones (AHLs) which are synthesized by the protein synthases (LuxI). The synthesized signaling molecules are secreted out of the cell and bind with receptor proteins (LuxR) of neighboring bacterial and decrease or increase the rate of transcription in target genes. The effect of the 66 AHL-degrading rhizobacteria (quencher) on modulation of LuxI/LuxR activity and inhibition of AHL synthesis was determined using an agar diffusion double streak assay and the Chromobacterium violaceum CV026 biosensor system. The isolate Pectobacterium atrosepticum SRI1043 used as AHL-producer. To test for potential LuxI inhibition, the AHL–producer isolate was placed in close proximity to the test quencher bacteria and the AHL biosensor strain placed distantly. In order to test for LuxR inhibition, the location of the AHL–producer and biosensor strains was reversed. Both LuxI and LuxR modulation was observed in 21 isolates. These isolates were identified as genera Bacillus, Brevundimonas, Citrobacter, Bordetella, Acinetobacter, Stenotrophomonas, Pseudomonas and Escherichia using biochemical tests and 16s rDNA sequencing. Bioassay for interference of AHL-dependent gene regulation in P. atrosepticum performed on Skim Milk Agar medium and antagonistic bacteria cultured near the pathogen and the reduction of protease production by this pathogen was evaluated by the reduction of clear zone in the agar. Five isolates reduced the protease production significantly.