Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI17

Abstract

The conjugative plasmid pYI17 (57.5 kb) isolated from Enterococcus faecalis YI717 confers a pheromone response on the host and encodes the bacteriocin 31 gene. Bacteriocin 31 is active against E. hirae 9790, E. faecium, and Listeria monocytogenes. pYI17 was mapped physically by restriction enzyme analysis and the relational clone method. Deletion mutant and sequence analyses of the EcoRI fragment B cloned from pYI17 revealed that a 1.0-kb 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 3.37 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 acid bacteria. Analysis of Tn5 insertion mutants and the resulting transcripts indicated that these genes are transcribed as an operon composed of bacA, bacB, and an open reading frame located downstream of bacB designated ORF3.Bacteriocins are produced by a wide variety of gram-positive and gram-negative bacteria. Bacteriocins are bacterial proteins which inhibit the growth of other bacteria closely related to the producer strain. They usually exhibit a relatively narrow spectrum of activity. Bacteriocins are thought to provide the producer strain with a selective advantage over other strains and are a factor in bacterial virulence. Research related to the gram-positive bacterial bacteriocins now appears to be centered on the bacteriocin activities of the lactic acid bacteria, and these bacteriocins were recently reviewed (24). Many of these strains are food grade organisms that are already widely used in the food industry, but recent research has indicated that these strains may find novel applications in food preservation. It is known that many Enterococcus faecalis strains produce bacteriocins (4, 7). The bacteriocin phenotype is frequently associated with the pheromone-responding conjugative plasmid of E. faecalis. These conjugative plasmids transfer at a high frequency in broth mating, a phenomenon related to their response to the specific peptide sex pheromones secreted by potential recipients (7-9, 14, 39, 40). The sex pheromone induces the synthesis of a surface aggregation substance that facilitates the formation of a mating aggregate (7-9, 14, 36).To date, only a few of the E. faecalis bacteriocins have been genetically and biochemically characterized. These include the hemolysin/bacteriocin and the peptide antibiotic AS-48, which are encoded by the E. faecalis conjugative plasmids pAD1 (58 kb) (8,9,11,20,43) and pMB2 (33), respectively. pAD1 and pMB2 were originally derived from E. faecalis subsp. zymogenes DS16 (43) and E. faecalis subsp. liquefaciens...

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…However, several class IIa bacteriocins, for example, listeriocin 743A (Kalmokoff et al 2001), bacteriocin 31 (Tomita et al 1996) and enterocin P (Cintas et al 1997) have been found to have a sectype leader. These bacteriocins are assumed to be secreted by sec-dependent exporting pathway.…”

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

“…N-terminal extensions of most lantibiotics and nonlantibiotics are of the so-called doubleglycine type (leader sequence) and are cleaved off concomitantly with export across the cytoplasmatic membrane by dedicated adenosine triphosphate-binding cassette transporters (ABC-transporters) and their accesory proteins Venema et al 1995). However, some class II bacteriocins, such as acidocin B (Leer et al 1995), divergicin A (Worobo et al 1995), bacteriocin 31 (Tomita et al 1996), enterocin P (EntP) , lactoccin 972 , propionicin T1 (Faye et al 2000), and enterolysin A (Nilsen et al 2003), contain N-terminal extensions of the so-called sec-type (signal peptide), which are proteolytically cleaved concomitantly with bacteriocin externalization by the general secretory pathway or sec-dependent pathway (van Wely et al 2001;Herranz and Driessen 2005). Several bacteriocins produced by enterococci, such as enterocins L50 (L50A and L50B), enterocin Q, and enterocin EJ97 (Cintas et al 1998(Cintas et al , 2000Sánchez-Hidalgo et al 2003), have been shown to be synthesized without N-terminal leader sequences, and may represent a new class of bacteriocins with a novel secretion mechanism.…”

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)

“…E. faecalis FA-2-2 and OG1-10 and E. hirae have been chosen as representative enterococcal strains for the examination and classification of the bacteriocins produced by the clinical isolates in this study. Class 1 types produce the ␤-hemolysin/ bacteriocin (cytolysin) and are active against a wide variety of gram-positive bacteria, including S. aureus (2,15,17,24,46). The ␤-hemolysin/bacteriocin (cytolysin) of pAD1 belongs to class 1.…”

“…They usually exhibit a relatively narrow spectrum of activity and are produced by a wide variety of grampositive and gram-negative bacteria (27). Bacteriocin production is thought to provide the host strain with an ecological or other selective advantage over other strains.Many Enterococcus faecalis clinical isolates produce a bacteriocin (3, 5), and the bacteriocin is frequently encoded on the E. faecalis pheromone-responding conjugative plasmid (6,14,21,46). Several E. faecalis bacteriocins have been genetically and biochemically characterized (15, 35), including the ␤-hemolysin/bacteriocin (cytolysin) (6,7,18,20,22) and the peptide antibiotics AS-48 (33), bacteriocin 21 (47), and bacteriocin 31 (46), which are encoded by the E. faecalis conjugative plasmids pAD1 (58 kbp), pMB2 (58 kbp), pPD1 (59 kbp), and pYI17 (57.5 kbp), respectively.…”

“…Several E. faecalis bacteriocins have been genetically and biochemically characterized (15,35), including the ␤-hemolysin/bacteriocin (cytolysin) (6,7,18,20,22) and the peptide antibiotics AS-48 (33), bacteriocin 21 (47), and bacteriocin 31 (46), which are encoded by the E. faecalis conjugative plasmids pAD1 (58 kbp), pMB2 (58 kbp), pPD1 (59 kbp), and pYI17 (57.5 kbp), respectively. A significant number of E. faecalis clinical isolates produce hemolysin/bacteriocin (10,26), and more than 50% of the hemolytic clinical isolates carry transferable hemolysin/bacteriocin determinants (21,26).…”