Mining Some Novel Bacteriocins In Bacillus Cereus Group

The discovery and application of antibiotics have made great contribution for human health.Howover,the abuse of antibiotics casued the rapidly increasing prevalence of multidrug-resistant bacterial strains.Now,the use of antibiotic have been rigorously controlled,and mining new antimicrobial drugs to treat infections has become urgent.Bacteriocins,which have many properties,are considered potential alternatives to conventional antibiotics.These include their potency(in vitro and in vivo),their low toxicity,the availability of both broad-and narrow-spectrum peptides,no easy to produce the cross-resistant with other antibiotics and these peptides can be bioengineered.In addition,with the rapid economic development and the improvement of people's living standards,human health and food safety have received increasing attention.Since some chemical preservatives are harmful to human health,mining natural food preservativs has been researched as a popular topic in the food industry.Since nisin successfully used as the preservative in food industry,bacteriocins was considered as a safe food preservative,and mining more bacteriocins which have excellent characteristic will provide a better service to hunman.So far,research on bacteriocins has focused primarily on lactic acid bacteria,such as Lactobacillus spp.and Pediococcus spp.,and the identified bacteriocins have more than one hundred.Members of the genus Bacillus cereus group are Gram-positive,endospore forming,closer relationship bacteria,and are also considered good producers of antimicrobial substances,and some bacteriocins have been identified in this group and some of them have good applition.For example,thuricin CD can kill a wide range of clinical C.diffcile isolates and reduce collateral impact on the commensal flora,and it may become a new drug to treat the infection caused by C.difficile.The comprehensive study obout the classification,distribution,and diversity of the bacteriocin gene clusters in the B.cereus group strains is still lacking.In view of the importance of bacteriocins and the great potential of the bacteriocin study in the B.cereus group,the purpose of this study is mining some novel bacteriocins which have better biochemical properties in Bacillus cereus group,and improves the understanding of the classification,distribution,and diversity of the bacteriocin gene clusters in this group strains.The conclusions of this study are as follows:1.B.cereus group have 6 types of bacteriocin biosythesis gene clusters.Genomes analysis of 225 strains of B.cereus group,we identified a large number of putative bacteriocin biosythesis gene clusters,and they involved in the biosythesis of 6 type of bacteriocins:lantibiotics,linear azole-containing peptides,head-to-tail cyclized peptides,sactipeptides,lasso peptides,and glycocins.We focused on two types of bacteriocins(lantibiotics and sactipeptides)which have great significance in application and did intensive studies on them.2.The B cereus group is an excellent reservoir of novel lanthipeptides.Lantibiotics are broad-spectrum antimicrobial subtances that can inhibit many Gram-positive clinical pathogens,including some multidrug-resistant ones,and mining novel lantibiotics may provide a new strategy for treatment of diseases caused by these organisms.So far,research on lantibiotics has focused primarily on lactic acid bacteria and other Firmicutes species.In this study,we identified 104 putative lanthipeptide gene clusters from 80 out of 225 strains of this group,and these gene clusters were further classified into 20 types according to their gene organization and the homologies of their functional genes.Among them,18 types were novel and have not yet been experimentally verified.Two novel lantibiotics(thuricin 4A-4 and its derivative,thuricin 4A-4D)were identified in the type 1-1 lanthipeptide gene cluster and showed activity against all tested Gram-positive bacteria.This work not only improves the understanding of the classification,distribution,and diversity of the lanthipeptide gene cluster in the B.cereus group but also provides a new means to quickly discover many more novel lantibiotics.Based on this research,we identified two novel lantibiotics which have applitions in food and pharmaceutical industry,respectively.(1)Three novel lantibiotics,ticins Al,A3,and A4,have extremely stable properties and are promising food biopreservatives.Nisin has successfully been used as a food preservative in over 50 conuntries,but its major drawback is its limited stability under neutral and alkaline pH conditions.To identify alternatives with better biochemical properties,we screened more than 100 strains of the B.cereus group.Three novel lantibiotics,ticins A1,A3,and A4,which were highly thermostable(121'C for 30 min)and extremely pH tolerant(pH 2.0 to 9.0),were identified in B.thuringiensis BhMB3201.They all showed potent antimicrobial activities against all tested Gram-positive bacteria and greater activities than those of nisin A against B.cereus and L.monocytogenes,two important foodborne pathogens.These three novel lantibiotics,with their extremely stable properties and potent antimicrobial activities,have the potential for use as biopreservatives.(2)Identifition of a novel two-component lantibiotic,thusin,which has potent antimicrobial activity against several Gram-positive pathogens.Lantibiotics,especially two-component lantibiotics,exhibit potent antimicrobial activity against some clinical Gram-positive pathogens and are considered potential alternatives to conventional antibiotics.In this research,we characterized a novel two-component lantibiotic termed thusin that consists of Thsa,Ths? and Ths?'(mutation of Ths?,A14G)was isolated from a B.thuringiensis strain BGSC 4BT1.Thsa and Ths?(or Ths?')exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells,and they were all highly thermostable(100? for 30 min)and pH tolerant(pH 2.0 to 9.0).Thusin shows remarkable efficacy against all tested Gram-positive bacteria and demonstrates 4-fold to 16-fold higher activity against various bacterial pathogens(B.cereus,L.monocytogenes,S.aureus(MRSA),S.sciuri,E.faecalis,and S.pneumoniae)than vancomycin,which had been used nearly 60 years to treat a number of Gram-positive bacterial infections.Moreover,thusin is also able to inhibit the outgrowth of the B.cereus spores.Given the potent antimicrobial activity of thusin against some Gram-positive pathogens,it has the potential for the development of new drug.3.Thuricin 67,a novel bacteriocin with a narrow spectrum of activity against S.pnerutmoniae and E.faecalis.Broad-spectrum antibitics have made great contributations for human against pathogens,but they also brought some negative consequences.They can inhibit or kill pathogenic bacteria,but also easy to damage the normal intestinal flora and cause varying degrees of dysbacteriosis.One possible means of avoiding such"collateral damage" would involve the application of a narrow-spectrum antimicrobials,such as the reported sactipeptide,thuricin CD.The purpose this study is mining some novel sactipeptides which have narrow-spectrum antimicrobial activity against some pethogens.In B.thuringiensis strain BGSC 4CE1,we characterized a novel sactipeptide termed thusin(4349.93 Da).It shows activity against S.pneumoniae and E.faecalis,but has no impact on most other Gram-positive bacterial and all tested Gram-negative bacterial.In addition,we screened 10 thuricin 67 resistant mutant strains and peformed the whole genome sequencing,to identify the target(receptor)of this bacteriocin on the sensitive strains.Given the specific antimicrobial activity of thuricin 67,it has the potential for the development of new drug that against the infections caused by S.pneumoniae and E.faecalis.In this research,we identified four novel bacteriocins which have applitions in food or pharmaceutical industry,offered a new understanding of the classification,distribution,and diversity of the bacteriocin gene clusters in the B.cereus group through the genome analysis,and also provied the important basis for mining more novel valuable bacteriocins.