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J. Bacteriol. doi:10.1128/JB.01056-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cloning and Genetic Analyses of the Bacteriocin 41 Determinant Encoded on the Enterococcus faecalis Pheromone-Responsive Conjugative Plasmid pYI14: a Novel Bacteriocin Complemented by Two Extracellular Components (Lysin and Activator)

Department of Bacteriology and Bacterial Infection Control and Laboratory of Bacterial Drug Resistance, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan

^* To whom correspondence should be addressed. Email: yasuike{at}med.gunma-u.ac.jp.

	Abstract

The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed it consisted of EcoRI fragments A to P. The clone pHT1100 containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp) conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6 kbp region within the EcoRI-AH fragments. Six ORFs were identified in this region and were designated ORF7 (bacL₁) ORF8 (bacL₂), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL₁, bacL₂, bacA and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL₁/L₂ and bacA mutants. bacL₁/L₂ and bacA encoded the bacteriocin component L and the activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL₁ protein had significant homology with lytic-enzymes that attack the Gram-positive bacterial cell wall. Sequence data for the deduced bacL₁ protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity and a third domain with a three-repeat structure directing the pro-enzyme to its cell surface receptor.