| PLNC8?/?,a Class IIb lactic acid bacteria bacteriocin,is a mixture of two-peptide PLNC8? and PLNC8? in equimolar ratio that inhibit both gram-positive and negative bacteria.Because of its broad inhibition spectrum,PLNC8?/? has a wide application prospect in the fields of food preservation.However,at present,the research on the antibacterial mechanism of PLNC8?/? is still in its infancy,which hinders its further commercial application.Therefore,in this paper,the PLNC8?/? of solid phase synthesis is used to study its inhibition spectrum and to explore the antibacterial mechanism of double targets in the cell membrane and genomic DNA of Micrococcus luteus(M.luteus).The details are as follows: 1.Determination of PLNC8?/? inhibition spectrum and minimum inhibitory concentration(MIC)of M.luteusPLNC8?/? with purity above 98% were synthesized by solid-phase synthesis,and it was found that PLNC8?/? could inhibit Gram-positive bacteria such as M.luteus,Staphylococcus aureus,Erwinia persicina and Lactobacillus plantarum,Gram-negative bacteria such as Salmonella and Vibrio parahaemolyticus,and fungi such as Rhodotorula rubra and Saccharomyces cerevisiae.Particularly,PLNC8?/? have the highest activity against M.luteus,and the MIC was 0.8 ?mol/L.2.Study on the mode of action of PLNC8?/? targeting in M.luteus cell membraneAnalysis of genomic DNA interaction between PLNC8?/? and M.luteus,it was found that the transmembrane electrical potential(??)and pH gradient(?pH)of the M.luteus were rapidly dissipated.And the ATP synthesis in the cells were inhibited after M.luteus was treated with 2 × MIC PLNC8?/?,while the electric conductivity increased.It could be observed that the surface and section morphology of M.luteus treated by 2 × MIC PLNC8?/? were significantly changed compared with that of untreated cells by scanning and transmission electron microscopy.Through the membrane damage rate and transmembrane efficiency test,it was confirmed that PLNC8?/? can destroy M.luteus cell membrane and has the activity of perforation.higher the concentration of PLNC8?/? could lead to the worse the cell membrane integrity of M.luteus.3.Study on the mode of action of PLNC8?/? targeting in M.luteus genomic DNAThrough the analysis of genomic DNA interaction between PLNC8?/? and M.luteus,it is found that PLNC8?/? can embed into M.luteus genomic DNA.Further through flow cytometry analysis,it was found that PLNC8?/? could affect DNA replication of M.luteus.The DNA replication related genes of PLNC8?/? could be analyzed by magnetic bead coupling method and qPCR method.It was found that PLNC8?/? can bind to dnaA,ligB and rnaseH sequences involved in DNA replication,and significantly lower their expression levels(p < 0.05).The above results showed that genomic DNA was one of the targets of PLNC8?/? acting against M.luteus.PLNC8?/? could embed into dnaA,ligB and rnaseH gene sequences involved in DNA replication.So that PLNC8?/? played antimicrobial activity by affecting DNA replication.4.Construction of antibacterial mechanism model of PLNC8?/? dual targetThrough laser confocal localization test,it was found that PLNC8?/? appeared in cell membrane and cytoplasm at the same time.In summary,PLNC8?/? can act on both cell membranes and genomic DNA dual targets of M.luteus:(1)PLNC8?/? can act on the cell membranes of M.luteus,so that the proton-motive force(PMF)will collapse,(2)PLNC8?/? can penetrate the cell membrane and get into the cells.While the ligB,dnaA and rnaseH sequences of genes related to DNA replication were embedded,and their expression was inhibited.Eventually,PLNC8?/? has antibacterial activity against M.luteus by this dual-target machine. |
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