Study On The Antibacterial Mechanism Of Bacteriocin BM1157 Against Escherichia Coli And Staphylococcus Aureus

Bacteriocins are a class of polypeptides or proteins with antibacterial activity synthesized by bacterial ribosomes,which mainly inhibit homology or close relatives.In recent years,bacteriocins have been widely studied due to their antibacterial properties,safety,and resistance to resistance,in order to become natural food preservatives and antibiotic substitutes in the future.The bacteriocin BM1157,as a new type of bacteriocin discovered by the research group in L.crustorum MN047,has been found to have broad-spectrum antibacterial activity in previous studies.In this study,we expressed BM1157 heterologously in E.coli,and carried out purification identification,characterization of properties and mechanism,and preliminary study of its molecular mechanism.The main conclusions obtained are:(1)We used the pET-30a(+)plasmid as the expression vector and E.coli BL21(DE3)as the host bacteria to successfully express the bacteriocin BM1157 heterologously,and used the double-layer Oxford cup plate method to express the prepared The crude sample of BM1157 was tested for antibacterial ability,and it was found that it had good antibacterial ability.(2)After a series of purification steps of BM1157 crude sample by dialysis,ultrafiltration,AKTA purification system using cationic SPFF column,reversed-phase liquid chromatography,and after liquid quality identification,the coverage of the identified amino acid residues was 98.18 %,all sequences of BM1157 except the methionine encoded by the start codon are covered.And through Fourier Infrared Transform Spectroscopy,it is known that it has a certain ?-helix and ?-sheet turning angle structure.(3)BM1157 has extremely low cytotoxicity,and has good antibacterial activity against both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria E.coli.In order to explore the antibacterial mechanism of BM1157,the release of extracellular DNA and the measurement of membrane potential changes were subsequently carried out.The results showed that BM1157 had no damaging effect on the cell membranes of E.coli and Staphylococcus aureus.Subsequent fluorescence real-time quantitative PCR assays found that BM1157 inhibited E.coli carbohydrate metabolism,amino acid metabolism,ribose transport and membrane protein expression and other processes,and significantly reduced the gene expression level of transcription regulator RNA polymerase?factor.For Staphylococcus aureus,BM1157 can inhibit its peptidoglycan synthesis and teichoic acid biosynthesis,inhibit the process of DNA replication and repair,and inhibit the cell cycle.The results of DNA binding experiments show that BM1157 can bind to the DNA of Staphylococcus aureus,but has no effect on the DNA of E.coli.(4)In order to obtain the target information of the molecular mechanism of BM1157,we obtained BM1157 resistant strains of E.coli and S.aureus through mutation screening,and performed whole genome sequencing of two wild strains and two resistant strains and obtained four The full genome information of the strain(the full length of the full genome sequence of the wild strain of E.coli is 4750508 bp,the full length of the full genome sequence of the resistant strain of E.coli BM1157 is5108639 bp;the full length of the full genome sequence of the wild strain of S.aureus is 2777619 bp,The full-length genome sequence of the resistant strain of S.aureus BM1157 is 2777010 bp),and then a comparative genomic analysis will be performed on the genomes of wild strains and resistant strains of E.coli and S.aureus respectively.The analysis results show that BM1157 There are a large number of different genes(325)between resistant bacteria and wild strains.Based on the analysis of the functions of these genes,we believe that BM1157 may have multiple antibacterial mechanisms against E.coli.Unlike E.coli,S.aureus has only 6differential genes.After analysis,we believe that BM1157 may change the pathogenicity of S.aureus due to adh esion and ?-toxin,and its target of action may be flavonoid metabolism.Chalcone isomerase in the process and cytochrome oxidase assembly protein in the process of cell respiration.In summary,the bacteriocin BM1157 has good broad-spectrum antibacterial activity,and has extremely low cytotoxicity.It has multiple antibacterial mechanisms and is not easy to produce drug resistance.It is extremely useful in the application of natural food preservatives and antibiotic substitutes.Great potential value.