Regulation Mechanism Of LuxS/AI-2 Quorum Sensing System On Biofilm Formation Of Lactobacillus Paraplantarum L-ZS9

Biofilm is the natural lifestyle of most microorganisms,which helps them to survive under unfavorable environments.Biofilm formation is regulated by quorum sensing(QS)system.Most researches have been focused on biofilm regulation and removal of pathogentic bacteria.However,there has been little researches on related field of beneficial bacteria.Lactobacillus paraplantarum L-ZS9 is a strain originally isolated from fermented sausage,proved to produce class ?b bacteriocins and has anti-cancer activity.This strain has the potential to be used as probiotics and fermentation starter.In this study,the advantages and influencing factors of biofilm formed by L-ZS9 were investigated.And the regulation mechanism of LuxS/AI-2 QS on biofilm formation of L-ZS9 was predicted and analyzed by transcriptomics,proteomics and genetic engineering technology.The main research contents are as follows:The biofilm lifestyle of L-ZS9 showed better stress resistance than its planktonic counterpart.Low pH value,high bile salt concentration,pepsin and trypsin inhibited or impaired the biofilm.And AI-2 could promote the biofilm formation and even relieve the inhibition and impairment of pepsin and trypsin,suggesting that biofilm formation of L-ZS9 was regulated by AI-2.Whole-genome sequencing indicated that L-ZS9 harbors gene luxS,which was involved in producing AI-2.And L-ZS9 has all genes involved in AI-2 production and no gene sahH,suggesting that SAH metabolic pathway is unique in L-ZS9.Therefore,this strain has the molecular basis of AI-2 production.Reporter luminescence bioassay also showed that L-ZS9 has the ability to synthetize and produce AI-2.And AI-2 activity was higher in biofilm supernatant than planktonic supernatant,suggesting that biofilm formation correlated to AI-2.AI-2 activity detection showed that D-ribose inhibited AI-2 activity of L-ZS9.And D-ribose inhibited biofilm formation of L-ZS9 induced by AI-2.Two dimensional electrophoresis analysis identified 27 differentially expressed proteins,which were mainly related to transcription,translation and carbohydrate metabolism.In accordance with the biofilm assay,opposite to the result of differentially expressed proteins regulated by D-ribose,exogenous AI-2 influenced expression of genes tuf fba,gap,rpoN,pgm,rib and nfo,suggesting that AI-2 and D-ribose regulated the expression of these genes inversely.Overexpression of gene luxS could increase AI-2 activity and biofilm formation of L-ZS9.RNA-seq identified 35 genes differentially regulated?2 folds in lucS-pMG76e-L-ZS9.And iTRAQ identified 35 proteins differentially expressed?1.2 or?0.83 folds in luxS-pMG76e-L-ZS9.Bioinformatics analysis showed that luxS overexpression mainly regulated transporter and membrane related proteins,suggesting that luxS overexpression may regulate these proteins to help bacteria response and adapt to extracellular environment.In addition,luxS overexpression influenced expression level of AraC family,LacI family and PadR family,which are related to biofilm formation and QS system mechanism.Based on above prediction and analyzation,recombinant strains overexpressing predicted genes were constructed and their biofilm formation were investigated.The results showed that LuxS/AI-2 QS system may modulate biofilm formation of L-ZS9 by regulating gene rpoN,guf,tuf,fba,gap,pgm,nfo,rib and sugE.Overexpression of gene rpoN,guf,tuf,fba,gap and pgm promoted biofilm formation,and overexpression of nfo,rib and sugE inhibited biofilm formation.And rpoN may regulate the expression of gene guf,nfo,rib and sugE to modulate biofilm formation of L-ZS9.This study has been firstly focused on the regulation mechnism of LuxS/AI-2 QS on biofilm formation of beneficial Lactobacillus.It provides a theoretical basis for development of biofilm probiotics and has a certain reference value to further researches on regulation of AI-2 on intestinal microflora.