The Study On Mode Of Action Of Class ?b Bacteriocin Plantaricin NC8 And Efficient Heterologous Experssion Of Its Genes

Plantaricin NC8 is a class ?b LAB bacteriocin.It consists of two peptides PLNC8? and PLNC8?,whose genes are next to each other in the same operon.Optimal antimicrobial activity is required as the presence of both two peptides.It was identified with great potential as natural food bio-preservatives and antibiotic alternatives.Plantaricin NC8 produced from LAB strains via a complex auto-induction quorum-sensing mechanism and very difficult to be isolated and purified with low yield.Thus it will unfavorable to the market application.In addition,no extensive studies of inhibitory spectrum and mode of action have been conducted for this bacteriocin.In our study,first the physical and chemical properties and structure features of Plantaricin NC8 were analyzed by bioinformatics methods and the relationship between structures and functions has been predicted.Then Plantaricin NC8 was synthesized by solid-phase synthesis.Additionally,the synergistic effect of PLNC8? and PLNC8? has been studied and the inhibitory spectrum has been achieved.Furthermore,the mode of action of Plantaricin NC8 has been researched.Finally,the high-efficiency Plantaricin NC8-producing genetically engineered bacteria have been constructed.The main contents are as follows:(1)The bioinformatic analysis of PLNC8? and PLNC8?:PLNC8? and PLNC8? are composed of common amino acids.They are both cationic peptides and are amphipathic and PI values present alkaline.According to the space structure prediction,the segments of PLNC8?have the similar structure with the transmembrane segment 2 of nmda receptor nrl and the structure of the phospholipid bilayer transporters.For PLNC8?,the structure is like PlLN J which belongs to another class?b bacteriocin Plantaricin JK.(2)The inhibition spectrum and synergitic effect study of Plantaricin NC8:Plantaricin NC8 was synthesized by solid-phase synthesis(purity? 98%).According to the agar-well test and minimal inhibitory concentration(MIC)test,the inhibition spectrum has been achieved.Plantaricin NC8 can inhibit a series of gram-positive and-negative bacteria and some fungi.It showed the highest activity against Salmonella spp.PLNC8? alone showed higher activity than PLNC8a alone.But the molar ratio of 1:1 in combination resulted in the highest activity.Thus the synergistic action of PLNC8? and PLNC8? was preliminary confirmed.(3)The study of "cell membrane permeabilization" bactericidal mechanism of Plantaricin NC8:Salmonella enterica subsp.enterica CGMCC1.1552 is one of the common food-borne pathogenic bacteria and used as the indicator strain.The dissipation of transmembrane electrical potential(??)and transmembrane pH gradient(?pH),and rapidly intracellular ATP depletion occurred after the indicator strain treated with Plantaricin NC8 and subsequently the electric conductivity(EC)of environments increased sharply.It resulted in dissipation of the cell proton motive force(PMF).Scanning and transmission electron microscopy were further used to observe that the cell surface and section morphology of the indicator strain changed after Plantaricin NC8 treated.In conclusion,the data showed that Plantaricin NC8 induced the increase of membrane permeabilization,which resulted in intracellular ion leakage,electrolytes efflux and final the indicator strain death.(4)The study of combination of Plantaricin NC8 and Lipid ?:Lipid ?,a precursor in the bacterial cell wall synthesis which was the common target against bacteria for many bacteriocins,was synthesized.The response values change of the fluorescent membrane potential-sensitive probe DiSC2(5)was measured after the indicator strain treated with Plantaricin NC8 with or without excess additional Lipid ?.The results indicated that the excess additional Lipid ? did not compete with Lipid ? in the Salmonella enterica CGMCC1.1552 cells to combine with Plantaricin NC8.Thus,Lipid ? is not the target of Plantaricin NC8 for pore-formation.(5)The study of "genomic DNA binding" bactericidal mechanism of Plantaricin NC8:According to the gel retardation assay of the binding of Plantaricin NC8 to S.enterica's genomic DNA,it is found that Plantaricin NC8 can strongly bind to the genomic DNA which resulted in the increase molecular weight of Plantaricin NC8-genomic DNA complex molecules.Thus addition of Plantaricin NC8 altered the migration of the genomic DNA in agarose gels.The electrophoresis bands without towing means Plantaricin NC8 only bound to the genomic DNA rather than disrupting them.With the increase concentration of Plantaricin NC8,genomic DNA and Plantaricin NC8 in combination made EB dye cannot insert into DNA base pairs,which caused the DNA bands did not have fluorescence.Therefore,the mechanism might be that Plantaricin NC8 can bind to genomic DNA of the indicator strain through competing with EB dye to intercalate into the nucleic acids.Moreover,the maximum absorption wavelength of genomic DNA(A260)was decrease with the increasing concentration of Plantaricin NC8.It is hypochromic effect which means Plantaricin NC8 can bind to genomic DNA of indicator strains and also intercalate into the nucleic acids.Based on the results,it is demonstrated that there were at least two key mechanisms underlying the antimicrobial action of Plantaricin NC8 against S.enterica,including the "cell membrane permeabilization" and "genomic DNA binding".However,Lipid ?,the common targets for many bacteriocins against bacteria,was not the target for Plantaricin NC 8.(6)Efficiet heterologous expression of genes of Plantarcin NC8:PLNC8? and PLNC8? were successfully heterologously expressed in E.coli BL21(DE3)by genetic engineering and formed the His-tag fusion protein.Through the optimization of Plackett-Burman experiment and response surface method,the yield of purified Trx-(His)6-PLNC8? fusion proteins was around 16-20 mg per liter of culture and the yield of purified Trx-(His)6-PLNC8? fusion proteins was around 12-16 mg per liter of culture.The fusion proteins were purified by Ni-NTA spin-columns and cleaved by enterokinase.The yield of purified PLNC8? was around 2-2.5 mg/L,and the yield of PLNC8? was around 1.5-2 mg/L.The molecular masses of peptides were identified using Tricine-SDS-PAGE and MALDI-TOF-MS.According to antimicrobial activity tests,heterologously expressed Plantaricin NC8 had high activity with synergistic effects and could diffuse better than solid-phase synthesized Plantaricin NC8 in media.It laid the foundation for heterologous expression of Plantaricin NC8 by lactic acid bacteria genetic engineering bacteria.Heterologously expressed Plantaricin NC8 has low cost and high yield.If the isolation and purification can be further simplified,it will have a good market application prospect.