BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF ENTEROCIN-P, A NOVEL SEC-DEPENDENT BACTERIOCIN FROM ENTEROCOCCUS-FAECIUM P13 WITH A BROAD ANTIMICROBIAL SPECTRUM

Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited m ost of tested spoilage and food-borne gram-positive pathogenic bacteri a, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand expo sure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and l ong-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cati on-exchange, hydrophobic-interaction, and reverse-phase liquid chromat ography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region reve aled the presence of two consecutive open reading frames (ORFs). The f irst ORF encodes a 71-amino-acid protein containing a hydrophobic N-te rminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enter ocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 t o -1), resulting in the mature bacteriocin consisting of 44 amino acid s, and,vith a theoretical molecular weight of 4,493. A second ORF, enc oding a putative immunity protein composed of 88 amino acids,vith a ca lculated molecular weight of 9,886, was found immediately downstream o f the enterocin P structural gene. Enterocin P shows a strong antilist erial activity and has the consensus sequence found in the pediocin-li ke bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.