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)

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 that 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 open reading frames (ORFs) were identified in this region and designated ORF7 (bacL 1 ) ORF8 (bacL 2 ), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL 1 , bacL 2 , bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL 1 and -L 2 and bacA mutants. bacL 1 and -L 2 and bacA encoded bacteriocin component L and 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 bacL 1 -encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL 1 -encoded 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 proenzyme to its cell surface receptor.Bacteriocins are bacterial proteins or peptides which inhibit the growth of other bacteria that are closely related to the producer strain. 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. 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). The hemolysin/bacteriocin of pAD1 is associated with virulence in animal models (4,25,29), and this plasmid is considered to be a typical ...

Partial Diversity Generates Effector Immunity Specificity of the Bac41-Like Bacteriocins of Enterococcus faecalis Clinical Strains

“…In contrast, class III bacteriocins, which are also referred to as bacteriolysins, are heat-labile antimicrobial proteins showing enzymatic bactericidal activity (7,19,20). To date, the only enterococcal bacteriolysins to be identified are enterolysin A and bacteriocin 41 (Bac41) (21)(22)(23).…”

“…Therefore, Bac41 is a unique two-component bacteriolysin consisting of two effector proteins, BacL 1 and BacA. Again, BacI is the cognate immunity factor providing self-resistance to protect a Bac41-producing E. faecalis from Bac41 activity (21).…”

“…Bac41 was originally cloned from the pheromone-responsive plasmid pYI14 of the clinical strain E. faecalis YI714 (21). Like other common bacteriocins, the bactericidal activity of Bac41 has a narrow spectrum against E. faecalis but is not active against E. faecium, Enterococcus hirae, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, or Listeria monocytogenes (21,24).…”

“…Like other common bacteriocins, the bactericidal activity of Bac41 has a narrow spectrum against E. faecalis but is not active against E. faecium, Enterococcus hirae, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, or Listeria monocytogenes (21,24). The determinant genetic element of Bac41 consists of six open reading frames (ORFs), including the four characterized genes bacL 1 , bacL 2 , bacA, and bacI ( Fig.…”

“…The determinant genetic element of Bac41 consists of six open reading frames (ORFs), including the four characterized genes bacL 1 , bacL 2 , bacA, and bacI ( Fig. 1) (21). The Bac41 lytic system has a classical antimicrobial effector/immunity module acting in interbacterial interaction (25).…”