Identification And Characterization Of Antimicrobial Compounds Produced By Bacillus Sp. BH072

The ongoing increase of antibiotic-resistance among pathogenic bacteria reduces the efficacy of most conventional antimicrobial drugs and threatens global health care.Moreover,pathogen contamination causes not only food safety issues,but also huge economic losses.By bio-control methods,the development of novel antimicrobial alternatives and biopesticides is one of the needs to combat this problem.Among these diverse compounds,those produced by bacteria have been most successfully applied as agents in the fields of food,medicine and agriculture.Antimicrobials produced by members of the genus Bacillus have been shown to have a broad spectrum of antimicrobial activity against pathogenic microbes.Firstly,we isolated a Bacillus strain BH072 from a honey sample showed antifungal activity against mold.Based on morphological,biochemical,physiological tests,and analysis of 16 S rDNA sequence,the strain was identified to be a new subspecies of Bacillus sp..It had a broad spectrum of antifungal activity against various mold,such as Aspergillus niger,Pythium,and Botrytis cinerea.In order to explain the antifungal activity,genes hag,ituA and tasA were retrieved and analyzed by PCR and sequencing.Flagellin and iturin A of fermentation broth were isolated and identified by a combination of several purification methods.Subsequently,the yield of iturin A was improved by optimizing its medium components and fermentation conditions by RSM.After the optimization of iturin A production by RSM,the mass concentration was 52.21 mg/ml,the antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg,which was 46.8%higher than before.The study provides an important experimental basis for the industrial production of iturin A and the agricultural application of Bacillus sp.BH072.Afterwards,the genome of the strain BH072 was sequenced and it was identified to be a Bacillus amyloliquefaciens strain.Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools,novel antimicrobial compounds,either ribosomally-or non-ribosomally produced,of various Bacillales species can be predicted and classified.Here,we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species.Moreover,we highlight the potential of several knownand novel antimicrobials from various species of Bacillales.Our extended classification of antimicrobial compounds demonstrates that the genus Bacillales provides a rich source of novel antimicrobials that can now readily be tapped experimentally,since many new gene clusters are identified.By BAGEL3,the entire type II lantibiotic mersacidin operon is perfectly conserved in the genome of BH072.Unfortunately,the mersacidin product could not be detected directly from the culture of BH072.Therefore,we employ the production machinery of the class I lantibiotic nisin(composed of the dehydratase NisB,the cyclase NisC and the transporter NisT)with an additional C-terminal decarboxylase(GdmD)to generate,modify,and secrete biologically active class I and II hybrid lantibiotics consisting of the N-terminal region of nisin and the C-terminal region of mersacidin.After cleavage of the leader peptide from the prepeptides by trypsin,modified hybrid peptides containing the decarboxylated C-terminus were shown to have a more potent antimicrobial activity against Micrococcus flavus than the ones without C-terminal AviCys.The study investigates different strategies to discover antimicrobials from Bacillus species.Based on the results,Bacillus sp.BH072 has great potential as a biological pesticide in agricultural production.We also provide insights into the genome mining and this work may advance the knowledge required for developing novel antimicrobials for medical application.