Regulation Of LuxS/AI-2-dependent Quorum Sensing On Leuconostoc Citreum 37 Biofilm Formation

Leuconostoc citreum(L.citreum)is a lactic acid bacteria found in animal milk and fermented products.L.citreum 37 produces a thicker biofilm during its growth,which plays an important role in bacterial resistance and adhesion.This study explored the role of quorum sensing LuxS/AI-2 in the formation of L.citreum 37 biofilm.The main experimental results are as follows:(1)The biofilm formation,AI-2 synthesis and LuxS/AI-2 key gene expression levels of the biofilm strains were significantly higher than those of the planktonic strains(P<0.05).Therefore,LuxS/AI-2 may be involved in the regulation of L.citrem37 the formation of biofilm.(2)Based on complete genome sequencing,the LuxS/AI-2 system of L.citreum37 was analyzed.The results showed that the genes that synthesize AI-2 include Met K,DNMT1,pfs,luxS,mmu M,etc.Among them,the pfs and luxS genes played a major role;the signal pathways involved in biofilm and quorum sensing include carbon metabolism,energy metabolism,and amino acids.Metabolism and so on.(3)The pfs and luxS of L.citreum 37 were heterologously expressed in E.coli BL21,and the AI-2 content of recombinant E.coli BL21 was detected.The results showed that the AI-2 content of luxS recombinant E.coli was significantly higher than that of pfs recombinant E.coli and empty plasmid strains(P<0.01),so the luxS gene plays a key role in the synthesis of AI-2.(4)The luxS homologous overexpression strain of L.citrem 37 was constructed,and its optimal growth temperature was 33°C,and the optimal film-forming temperature was 37°C.Although the growth of overexpression strains lags slightly behind that of wild-type strains,the amount of AI-2 synthesis and biofilm formation in overexpression strains are significantly higher than those of wild-type strains(P<0.05).(5)Through physical and chemical experiments and microscopic examinations,the promoters(0.05% Fe Cl3)and inhibitors(9% D-ribose)that regulate the formation of strains' biofilm were screened out.The inhibitor was applied to L.citrem 37,and through differential protein analysis,it was clear that opp A,pel,cit E,pgm,GAPDH/gap A,GPI/pgi and other genes may be involved in the formation of L.citrem 37 biofilm.(6)The optimal immobilization time of luxS overexpression strain and wild-type strain on NCA(cellulose acetate nanofiber membrane)were optimized and determined to be 24 h and 20 h,respectively.The survival rate of immobilized luxS overexpression strains in simulated gastrointestinal fluid was higher than that of wildtype strains.And the wild-type L.citrem 37 and luxS overexpression strains fixed in NCA can ferment skim milk 5 times.This study clarified the regulation effect of LuxS/AI-2 on the formation of L.citrem 37 biofilm.A strain with thick biofilm and high AI-2 synthesis was obtained by constructing luxS homologous overexpression strain.It provides a theoretical basis for the development and application of high-activity coated probiotics,and provides a reference for the study of quorum sensing and biofilms of other probiotics.