GENETIC ANALYSIS OF THE BACTERIOCIN DETERMINANT ENCODED ON A CONJUGATIVE PLASMID PYI17 (57.5kb) OF ENTEROCOCCUS FAECALIS YI17

Abstract

The conjugative plasmid pYI17 (57.5kb) isolated from Enterococcus faecalis YI17 confers a pheromone-response on the host and encodes the bacteriocin 41 (Bac41) gene. Bac41 is active against E.hirae 9790, E.faecium and Listeria monocytogenes. pYI17 was physically mapped by restriction enzyme analysis and the relational clone method. Deletion mutants and sequence analyses of the EcoRI fragment B cloned from pYI17 revealed that a 1.0kb fragment contained the bacteriocin gene (bacA) and an immunity gene (bacB). This fragment induced bacteriocin activity in E.faecalis OG1X and E.hirae 9790. The bacA gene is located on the pYI17 physical map between 3.37kb and 3.57 kb, and bacB is located between 3.59 kb and 3.87 kb. bacA encodes 67 amino acids and bacB encodes 94 amino acids. The deduced amino acid sequence of the BacA protein contained a series of hydrophobic residues typical of a signal sequence at its amino terminus. The predicted mature BacA protein (43 amino acids) showed sequence homology with the membrane active class II bacteriocins of lactic acids bacteria (LAB). Analysis of Tn5 insertion mutants and their transcripts indicated that these genes are transcribed as an operon composed of bacA, bacB, and an open reading frame located downstream of bacB designated as ORF3.