Functional Study Of The Key Gene LuxS Of Lactobacillus Plantarum Quorum Sensing Signal Molecule Synthesis

Quorum sensing(QS)is a bacterial communication signalling system that allows bacteria cells to communicate with each other.Lactobacillus belongs to Gram-positive bacteria,possessing oligopeptide-mediated QS system and interspecies QS system mediated by AI-2.Studies shown that QS system affect the resistant to acid,cholesterol salt and antibacterial of lactic acid bacteria.The Lb.plantarum AY01 and YM-4-3 were isolated from fermented goat milk and Yimen Douchi respectively,and the analysis of AY01 and YM-4-3 genome found that AY01 harboring one luxS ORF while YM-4-3 contains two,the key gene of the interspecies QS system,which can express LuxS protein to produce QS signaling molecule AI-2.This report was focused on the knockout of luxS of AY01,and the difference of growth acidogenic,tolerance for acid and bile salt,biofilm-forming ability and ablity to inhibit the formation of biofilm of ECEC between AY01 and its luxS mutant.In addition,the prokaryotic expression vectors pET28a-luxSl and pET28a-luxS2 were constructed and expressed in E.coli BL21(DE3).Results for these tests are outlined below:(1)Temperature-sensitive knockout vector pKML05 was used in this study,and the luxS mutant of AY01 was built by homologous recombination,named LuxS△1.(2)Bioluminescence experiment of V.harveyi BB170 shown that LuxS△1 could not produce AI-2,indicated that the luxS plays key role in the AI-2 synthsis pathway of AY01.(3)There was no significant difference in growth,biofilm formation,acid and bile resistance.and biofilm-forming ability between AY01 and LuxS△1.(4)The adhesion of AY01 and LuxS△1 to Caco-2 cells showed that the adhesion ability of LuxS△1 cells cultured at 14 h and 27 h was much lower than that of AY01.which indicated that luxS played an important role in the adhesion of AY01 to Caco-2 cells effect.(5)Cultures of AYOI has stronger restraint to the biofilm formation of EHEC than LuxS△1.(6)The expression vectors pET28a-luxSl and pET28a-IuxS2 were constructed and the LuxSl and LuxS2 proteins with purity of more than 90%were successfully purified by optimized purification.(7)Both purified LuxS1 and LuxS2 could catalyze the synthesis of AI-2 by SRH.Al3+,Ca2+ and EDTA could enhance the enzyme activity of LuxS1,while Al3+,Ca2+,Fe2+,Mn2+ and EDTA can enhance the enzyme activity of LuxS2.Both LuxS1 and LuxS2 are strongly inhibited by Cu2+,Zn2+,Ni2+ and glycerol.