Construction Of Lactobacillus Paracasei HD1.7 PrcR, PrcK Gene Deletion Mutant

A lactic acid bacteria,called Lactobacillus paracasei HD1.7,was isolated from Chinese sauerkraut juice in 2003.Further research found that non-acid antimicrobial substance could be produced by this strain and was a peptide that was active against many other Gram-positive bacteria,Gram-negative bacteria,and Saccharomyces cerevisiae.At the same year,Jiro Nakayama et al.obtained a series of genes?prcA,prcK,prcR?from L.paracasei E93490 and these genes were defined as the putative three-component regulatory system components which regulated bacteriocin production?Quorum sensing of lactic acid bacteria was often regulated by three-component regulatory system composed of autoinducing peptide,sensor kinase and response regulator?.Accordingly,the peptide secreted by L.paracasei HD1.7 might be bacteriocin and L.paracasei HD1.7 also had the same situation as L.paracasei E93490.Until now,there was few knowledge about how the bacteriocin was produced by L.paracasei and how its production was regulated.So,this work will be very significant.First,according to the sequence of L.paracasei E93490 in NCBI,the presences of prcK and prcA in L.paracasei HD1.7 were examined by PCR.The results showed that the length of prcK was 1299bp and the sequence was submitted into NCBI;the length of prcA was 138bp and the sequence was submitted into NCBI.The information of prcK and prcA were revealed and forecasted by bioinformatics method,which found the relationships between prcK and sensor kinase and between prcA and response regulator.It provided basis for sequent research on functions of the putative quorum sensing related genes.Second,gene knock-out technology was used to explore the function of prcK and prcR.On the basis of plasmid pUC18 and p2NIL,suicide plasmids,pYTKLKRT,pYTKA,pYTRLRRT,p YTRT,and pYTKRT,were constructed to get mutant strains of prcK deletion and prcR deletion,among which p YTKLKRT and pYTKA were used for deleting prcK,and pYTRLRRT and pYTRT were used for deleting prcR,and pYTKRT was used for deleting both prcK and prcR.Electrotransformation technique was used to transform plasmids into L.paracasei HD1.7.The optimal electrotransformation conditions for L.paracasei HD1.7 were explored here.The result showed a high electrotransformation efficiency could be obtained.Recipient cells were first treated by1%of glycine.When the OD₆₀₀ reached 0.5-0.6,cells were washed by electrotransformation buffer of AEB1,as well as the 9kv/cm of field strength and one time of pulse.Then MRS culture media was added quickly into cells and cells were recovered 2-3h.These suicide plasmids were imported to L.paracasei HD1.7 by electrotransformation technique.The mutant strains were screened by PCR.The results showed that these plasmids were successfully imported into L.paracasei HD1.7 and homologous recombination definitely occurred at the specific place.Antibacterial tests were done to observe antibacterial condition of mutant strains.The results showed that the antibacterial activities of mutant strains were weaker than the original strain and the titer of antimicrobial peptide of prcK knockout mutant strain,prcR knockout mutant strain and prcKprcR knockout mutant strain was 1538.29AU/ml,1572.26AU/ml and1276.53AU/ml respectively and was 23.61%,21.93%and 36.61%lower than the titer of the original strain respectively,which testified that the deletion of prcK and prcR had an influence on production of bacteriocin.Taking into account all the results,the work could testify that prcK and prcR was sensor kinase and response regulatorBy constructing prcK knockout mutant strain,prcR knockout mutant and prcKprcR knockout mutant strain,the research could be a potential basis for the exploration of prcK,prcR functions and L.paracasei quorum-sensing system and the mechanism of antimicrobial peptide production,which could ask people to change the regulation process of antimicrobial peptide production in certain direction and then to construct antimicrobial peptide high-production strain that could highly express large amounts of antimicrobial peptide in a low number of strains.It provided an example for quorum-sensing theoretically,and could be the theoretical basis for industrial production of antimicrobial peptide.