The Diversity And Inhibition Activity Of The Enterolysin And Helveticin J Gene From Chinese Traditional Foods

Expansion of food industry and international trade in food stuffs, together withincreasing worldwide attention to food safety heightened the need of exploring naturalpreservatives such as bacteriocins to prevent food contamination and spoilage.Bacteriocins produced by lactic acid bacteria (LAB) are a heterogeneous group ofactivated peptide or proteinous material，most of which are provided with inhibition forbacteria in near relation,and the minority with wider bacteriostatic spectra．Bacteriocinsfrom LAB are currently classified into three different major classes. Class I bacteriocins,the lantibiotics, are small peptides that undergo extensive posttranslationalmodifications. Class II bacteriocins are unmodified, heat-stable peptides. Class IIIbacteriocins, the least well characterized so far, are large and heat-labile antimicrobialproteins. Large bacteriocins are produced by many members of the Archaea andBacteria. To our knowledge, the bacteriolysins were the least investigated bacteriocins,with only five members have been characterized genetically to date. They wereMillericin, Enterolysin A (EnlA), Zoocin A, Lysostaphin and Helveticin J (HlvJ),produced by Streptococcus milleri, Enterococcus faecalis, Streptococcus zooepidemicus4881, Staphylococcus aureus, and Lactobacillus helveticus, respectively, all of whichbelong to Lactobacillales. In this study, we study the Enterolysin A and Helveticin J.Enterolysin A is a heat-labile protein with a broad inhibitory spectrum, which breaksdown cell walls of sensitive bacteria. This bacteriocin was shown to display a broadlytic activity against several bacterial genera, e.g. Lactobacillus, Lactococcus,Pediococcus, Enterococcus, Bacillus, Listeria and Staphylococcus spp. Helveticin J is achromosomally encoded bacteriocin that has a narrow inhibitory spectrum and isproduced by Lactobacillus helveticus481. This bacteriocin is a37,511-Da heat-labileprotein with a bactericidal mode of action. There are many kinds of fermented foods made of traditional manufacturing proceduresfor hundreds or thousands of years in China, some kinds of fermented foods have beenassessed to possess a very diverse and rich microflora. Therefore, microbialcommunities of fermented foods could serve as a gene pool coding for bacteriocins, thepotential of which, however, has largely been untapped. In this study, we employed aculture-independent metagenomic approach to investigate the bacteriolysins in six kindsof popular traditional fermented foods in China.In this study, we uncovered a significant diversity of enterolysin A and helveticin J genesequences belonging to bacteriolysins in popular Chinese traditional fermented foodsusing culture-indepe ndent metagenomic approach. The CODEHOPs targetingconserved motifs of the enterolysin A and helveti cin J genes from Lactobacillales weredesigned to amplify respective gene sequences from six metagenomic DNA extracts byPCR. The amplicons were evaluated by sequence clone libraries and phylogeneticanalyses. All translated enterolysin A sequences (77-80amino acids) derived from fourlibraries were grouped into one cluster with84-96%identity to homologs fromLactobacillus in Gen Bank data base. Deduced helveticin J sequences (169-196aminoacids) derived from five libraries were classified into two distinct clusters, whichshared58-100%identity with public homologs in the database. Based on the obtainedsequences, two of intact enlA and hlvJ genes was amplified by genome walking TAIL-PCR, and cloned in to the expression vector pET-28a in Escherichia coli BL21,respectively. The recombinant EnlA (DFE5) producing a30.6-kDa protein displayedantimicrobial activity against three bacterial genera, including human pathogensListeriamonocytogenes and Staphylococcu saureus. The expressed HlvJ (RS9) encodinga249-aa protein was observed to inhibit Lactobacillus helvetius. The recombinant EnlA(GCE11) producing a25.3-kDa protein displayed antimicrobial activity against threebacterial genera, including human pathogens Listeriamonocytogenes and Staphylococcu saureus. The recombinant HlvJ (RW21) producing a36.7-kDa protein displayedantimicrobial activity only against Lactobacillus helvetius. This study demonstrated thevalidity of t he metagenomic approach used in thi s study for identifying natural variantsdirectly from complex food samples, but also provided the evidence that Chinesetraditional fermented foods could be a valuable bacteriocin gene source for furtherexploration in food industry.