Regulatory Mechanism Of Quorum Sensing On L-amino Acid Oxidase In Pseudoalteromonas Sp.R3

Quorum-sensing?QS?bacteria produce and release signal molecules.When the signal increases with bacterial density and reach a critical concentration,the QS will alter the gene expression and subsequently regulate the biological behavior of bacteria,such as biofilm formation,antibiotic production,bioluminescence and toxin production.L-Amino acid oxidase?LAAO?can oxidize the L-amino acids to generate H₂O₂ which cause oxidative stress.Recently,LAAO-producing Pseudoalteromonas sp.R3 which can cause QS-biosensor discolor was isolated from marine niches.It has been reported that QS can regulate the genes associated with ROS scavening system.This study aims to study regulatory mechanism of quorum sensing on LAAO synthesis in strain R3.First,we sequenced the draft genome of strain R3.The draft genome is estimated to 5.58 Mb,with a GC content of 47.63%and 5429 putative ORFs,with an average size of 944 bp.Approximately 87.8%of nucleotides were predicted to be ORFs.The analysis show that strain R3 has one acyl-homoserine lactone synthetase gene ahl,seven LuxR family transcriptional regulator,QseC/QseB,QseE/QseF and AI-2 system.Then,we confirmed strain R3 can produce acyl homoserine lactone as signal molecule through different methods.We detected the cell density,signal molecules and LAAO activity during the static culture and used real-time fluorescent PCR to analyze the transcription level of ahl and lao.The results showed that the higher the cell density is,the more AHLs generated and the higher the LAAO is.Besides,there is a positive correlation between the expression of ahl and lao.At last,the suicide plasmid pCVD442/Em-ahl in donor E.coli S17-1????was transferred into the recipient Pseudoalteromonas sp.R3 for the generation of ahl gene deletion strain?QS mutant strain?.Again,the cell density,signal molecules and LAAO activity during the static culture and the transcription level of ahl and lao were analyzed in strain R3 and QS null mutant to reveal the regulation mechanism of QS on LAAO synthesis.