Research On Regulation Mechanism Of Genes Related Cell Wall In Lactococcus Lactis Under Acid Stress

Lactococcus lactis F44 produces a large amount of lactic acid during the growth,acidifying the fermentation broth,which will inhibit the growth of the strain and reduce the production of Nisin.The cell wall serves as the first barrier for bacteria to resist environmental pressures and has an important protective effect.Our previous research found that over-expression of some genes related to the cell wall synthesis and modification could improve the acid tolerance of strain and the production of Nisin.Acid stress can induce some transcription factors to activate or inhibit the expression of some genes,thereby regulating intracellular metabolic pathways.The research on transcription factors regulating cell wall will lead a foundation for the comprehensive analysis of regulation mechanism in response to acid stress,which is of great significance for improving the tolerance of strains and constructing high-yield lactic acid bacteria.In this study,based on the proteome of L.lactis F44 under acid stress,nine genes related to the cell wall were selected as candidate genes to construct homologous overexpressed strains Fyid C,Fylb A,FdacA,FdacB,FdltD,Ferfk,Fhrt A,FWP?017864383.1 and FWP?080613266.1.Fermentation and tolerance experiments were carried out,Overexpressing dacB,dltD and erfk could improve the growth,and the survival rate of strain under acid or salt stress,which were selected to further investigate.dacA and dacB are genes encoding D,D-carboxypeptidase and L,Dcarboxypeptidase,respectively,but they showed completely different results in the proteome and tolerance experiments,which implied that they might play different roles in the regulation of cell wall and need to be further studied.Therefore,the DNA affinity pull-down experiment was performed to identify the transcription factors combined with the promoter regions of dacA,dacB,dltD,and erfk.The results showed that RcfB could bind to the promoter regions of the dacA,dltD and erfk;no transcription factor could bind to the promoter region of the dacB;the twocomponent system transcription factor Tcs R7 could bind to the promoter region of dltD.The interactions between the transcription factors and target genes obtained by the DNA affinity pull-down experiment was verified in vitro by EMSA.The results showed that the Tcs R7 protein purified in vitro could bind to the promoter region of dltD,but RcfB protein could not bind to promoter regions of dacA,dltD and erfk.In addition,to further determine the regulation between them,real-time PCR technology was used to analyze the regulatory effect of transcription factors on the cell wall-related genes.The study found that under acidic conditions,the expression of tcs R7 increased,which could promote the expression of the dltoperon.It was indicated that under acid stress,the transcription factor Tcs R7 can increase the positive charge of the cell surface and increase the acid tolerance of strain by directly up-regulating the expression of the dltoperon.Also,there was no significant regulation between the transcription factor RcfB and dacA,dltD and erfk gene detected by qRT-PCR.