Mining Bacteriocin Gene,Molecular Modification And Their Synthetic Regulation Mechanism In Lactobacillus

The widespread use of antibiotics in medicine,animal husbandry,and agriculture has increased the antibiotic resistance.Resistant and multi-drug resistant bacteria,such as methicillin-resistant Staphylococcus,vancomycin-resistant enterococci,extended-spectrum?-lactamase-producing Escherichia coli and Klebsiella pneumoniae,carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii have put us in a situation where no antibiotics are available.How to reduce the increasing speed of antibiotic resistance and reduce the residual of antibiotics in animal products is related to everyone's health.Researchers have been looking for and designing new antimicrobials to solve antibiotic resistance and antibiotic residues.Bacteriocin is a peptide or peptide derivative of an antibacterial activity synthesized by bacteria,which are high efficiency,no residue,and not susceptible to drug resistance,and has become a potential substitute for antibiotics.In this study,we aim to get new,efficient,and broad-spectrum bacteriocin by bioinformatics based on the genome of lactic acid bacteria.We also design,modify,bacteriocin based on the relationship of structure and function to improve their antibacterial activity and inhibition spectrum.Finally,we analyzed the regulation mechanism of plantaricin synthesis by molecular biology methods for increasing the yield of plantaricin.1.Analysis of bacteriocin in lactic acid bacteria by bioinformatics methodsWe analyzed the bacteriocin gene in two aspects by analyzing the data of the genome data of Lactobacillus plantarum 163 and Lb.helveticus NX2-6.On the one hand,we predicted the bacteriocin that the lactic acid bacteria can synthesize by analyzing the bacteriocin clusters.The results indicated that the Lb.plantarum 163 has a plantaricin-related gene cluster "pln".Then we got plantaricin E,Em,F and A in the cluster by chemically synthesized and heterologous expression.The results of antimicrobial activity showed that the activity of plnEm was significantly lower than that of the wild-type plnE.Among these plantaricin,plnA has the highest antibacterial activity and the broadest antibacterial spectrum,and the minimum inhibitory concentrations against Escherichia coli and Staphylococcus aureus are 25 ?g/mL and 50 ?g/mL,respectively.On the other hand,we dig out potential bacteriocin that is not in the gene cluster.A novel plantaricin plnl was obtained in Lb.plantarum 163,which had antibacterial activity against Gram-positive bacteria and no activity against Gram-negative bacteria.The spatial structure prediction results show that it has a similar spatial structure to plantaricin EF,and both are composed of ?-helix and have a positive charge.The results of antimicrobial mechanism indicated that plnl increased the permeability of cell membranes,leading to intracellular material leakage,but it does not cause cell death at lower concentrations.Besides,we have mined two analogues of Helveticin J(NX267 and NX371)from Lb.helveticus NX2-6,which monomers have no activity against bacteria,yet multimers have activity against gram-negative and-positive bacteria.Helveticin NX267 and NX371 bound to peptidoglycan layer and hydrolyze peptidoglycan such as lysozyme.2.Modification of plnA to improve its antibacterial activity and outer membrane permeabilityAccording to the AMP-membrane model,the model of AMP-outer membrane of Gram-negative bacteria,a series of mutants of PlnA were obtained by changing its amino acid,charge number,hydrophobicity and structure.The antimicrobial activity showed that the antimicrobial activity significantly increased when increasing the positive charge number and the hydrophobicity.Among the various mutants,antimicrobial activity of OP4 increases of 6.25 times and an average MIC for various Gram-negative bacteria is 4 ?g/mL.In addition,PlnA and its mutants increase the permeability of the outer and inner membranes,leading to leakage of intracellular material.Compared with the permeability of the inner membrane,OP4 has a stronger outer membrane permeability,and significantly improve the outer membrane permeability of E.coli 0157 at 1/4 MIC(1.5 ?g/mL).Binding assays in vitro showed that the antimicrobial activity decreased and endotoxin levels decreased after mixing OP4 with LPS.The molecular weight of complex OP4-LPS is 73 KDa monitored by HPLC.Then we knocked out the synthesis gene(IpxL and waaC)of LPS and constructed the lipopolysaccharide-deficient mutant.The outer membrane permeability of mutant showed that the outer membrane permeability caused by OP4 decreased,and antibacterial activity of OP4 decreased.Experiments confirmed that OP4 binds to the lipopolysaccharide of the outer membrane,reduces the strength of the linkage between the lipopolysaccharides,interferes with the arrangement of the lipopolysaccharide layer,and increases the permeability of the outer membrane.As we know,the potency of antibiotics is closely related to the permeability of the outer membrane of the negative bacteria.In this chapter we use OP4 to increase the permeability of the outer membrane that significantly increased the potency of hydrophobic antibiotics against E.coli.For example,the potency of erythromycin increased by 62.5 times,the titer of tetracycline antibiotics increased by 8 times,and the titer of sulfa drugs increased by 16 times.In addition,OP4 also increased the potency and inhibition spectrum of the antibacterial lipopeptides.For example,for the potency against E.coli,Surfactin increased by 1000 times,Bacillomycin D increased by 250 times,and Fengycin increased by 62.5 times.The permeability of OP4 to the outer membrane barrier of negative bacteria is of great significance for improving the potency of existing antibiotics and solving the problem of antibiotic resistance of negative bacteria.3.Analysis of the regulation mechanism of plantaricin synthesisFirst,we performed cluster analysis on 20 plantaricin gene clusters to obtain four types of regulatory systems.Amino acid mutations were made to the autoinducer peptide(plnA)and histidine kinase(plnB)of Lb.planta rum 163.The N-terminal hydrophobic region(ISLM)of plnA activates plnB,and phosphorylates plnD by binding to the hydrophobic pocket(FASQF)of loop2 of plnB via a hydrophobic interaction.Unlike previous studies,our experiments confirmed that phosphorylated plnD activates the synthesis of plantaricin.In addition,we identified a new inducer,acetate,from the fermentation broth of Lb.plantarum 163.It binds to the positive charge pocket(RRYSHK)of loop4 of plnB by electrostatic interaction,which activates plnB to regulate the synthesis of plantaricin.The side chain of Phel43 at the N-terminus of loop4 plays an important role in maintaining positive charge pockets and specific recognition of acetate.The mode in which acetic acid activates plnB is also present in the other three type of plnB,indicating that acetate as an inducer is ubiquitous in Lb.plantarum.In vitro experiments confirmed that plnA and acetate are independent of the activation of plnB,and they have additive effects before the plnB activity reaches a maximum.Interestingly,when the concentration of one inducer was too high to activate plnB,another inducer could still activate plnB at a suitable concentration.Since plnA is synthesized in the logarithmic growth phase,and decreased in the stationary phase,yet acetic acid begins to synthesize in the stationary phase.Therefore,plnA and acetate have growth-specificity regulation mode in Lb.plantarum,which plnA plays a role in the logarithmic growth phase,and acetate plays a role in the stationary phase,and the two coordinately maintain the synthesis of plantaricin.In summary,we are committed to mining new bacteriocin from lactic acid bacteria or designing new bacteriocin to solve the antibiotic resistance.Increasing the production of bacteriocin by analyzing the synthesis mechanism of bacteriocin to reduces the cost of bacteriocin.In the end,we hope to reduce the use of antibiotics in food processing,medicine,animal feeding,etc.through our bacteriocin products,reduce the generation of antibiotic resistance,and reduce the residues of antibiotics in animal products.