| There is a large output of aquatic products in China,fish production accounts for about 60%of the total production of aquatic products.However,because of its abundant protein and moisture content,fish can easily be contaminated by microorganisms after fishery harveating,which greatly reduces its economic value.The quorum sensing of dominant spoilage bacteria of aquatic products is an important cause of quality deterioration,and the signal molecules secretion of quorum sensing such as N-acyl-homoserine lactones(AHLs)can regulate their virulence factor production through a combination of receptor proteins.Therefore,the quorum sensing provides an alternative strategy for the preservation of aquatic products.However,the current researches on quorum sensing are mainly focused on the isolation and identification of signal molecules producing strains and the AHLs detection in food.There is seldom research on the regulation system,especially the practical role of this system under natural conditions.In this study,Pseudomonas fluorescens(PF-08)which separated from spoilage turbot were used as test strain,and the inherent quorum sensing regulated genes were explored by whole genome sequencing technology.Then,the influence of different environmental factors on expression of quorum sensing regulated genes were teated.At the same time,the exogenous signal molecules were added to explore the exogenous signal molecules utilization of PF-08 through its own QS system.This study was aimed to clarify the spoilage mechanisms of fish caused by microorganism,and to provide theoretical basis for the development of new seafood preservation techniques based on the targeted inhibition of the spoilage bacteria underlying the QS systems.1.The PacBio RSII platform was used to sequence the whole genome of PF-08 which was isolated from the decayed turbot,and the important genetic information was obtained.The size of the genome is 6.03 Mb,and the ratio of GC is 61.6%,which consists of 5515 Genes,of which 4900 is the encoding sequence(CDS).According to the cluster analysis of COG,GO and KEGG,we found that the functional genes of strain PF-08 were mainly concentrated in biological metabolism and signal transduction,etc,which showed that the strain PF-08 had high activities in metabolic and environmental signal processing.The QS regulatory genes in PF-08 were identified as the signal molecular synthetase gene rhlI and the receptor protein gene rhlR,respectively,by NCBI Blast analysis.2.The expressions of rhlI and rhlR in PF-08,under different environmental factors,were detected by RT-qPCR and 16S rRNA was used as reference gene.The expression difference of related genes were calculated by ??Ct method.The results showed that the environmental factors could affect the expression of rhll and rhlR in PF-08.The expression of rhlI gene in different carbon source medium was lactose>sucrose>maltose>glucose>fructose>xylose,which rhlR gene was lactose>glucose>maltose>sucrose>fructose>xylose.The NaCl concentration in the environment was negatively correlated with the expression of rhlI and rhlR,and they could be well expressed in the environment of weak acid(pH=6)or low temperature(15?).And the expression of rhll and rhlR were inhibited under high temperature(37?)and alkaline environment(pH>7).3.The AHLs eavesdropping ability of PF-08 was studied through biofilm and extracellular protease experiments,and was verified by the results of GC-MS,RT-qPCR and molecular docking.The results showed that the production of biofilm and protease of strain PF-08 were enhanced by eavesdropping the signal molecules in the environment.The addition of exogenous signal molecules significantly increased the expression of AHLs receptor protein gene rhlR in PF-08,and the role of C14-HSL was the most obvious.The results of GC-MS showed that the content of C14-HSL decreased 82.5%after clutureing.In addition,the docking results also showed that C14-HSL was able to bind to the RhlR receptor protein as a natural ligand C4-HSL by hydrogen bonding. |
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