Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning

Abstract

Thirty-seven nonhemolytic/nonbacteriocinogenic mutations in Enterococcus (Streptococcus) faecalis plasmid pADi were generated by Tn917 insertion. All were found to belong to one of two complementation classes.Each class of mutants secreted either hemolysin/bacteriocin (Hly/Bac) component A or L into the culture medium. DNA encoding Hly/Bac was cloned in Escherichia coli in which both components of the hemolysin were expressed individually and collectively. The region encoding components A and L was further defined by deletion analysis and physically mapped. A total of approximately 8.4 kilobases of pADl DNA were observed to be required for hemolysin expression. Hly/Bac activity of the wild-type and the inactive L substance was observed to be heat stable. Active Ely/Bac resulting from incubating separately secreted components A and L was also found to be heat stable. The results indicate that component A activates component L and that activated component L possesses the Hly/Bac activity. Component A was also observed to be associated with host immunity to the Hly/Bac.In certain geographical locations as many as 60% of clinical Enterococcus (formerly Streptococcus) faecalis isolates produce a cytolytic toxin which lyses human, rabbit, and horse erythrocytes (20,23). This hemolysin also possesses bacteriolytic activity and mediates the lysis of a broad range of gram-positive bacteria (2, 4, 6). Epidemiological studies have shown that hemolysin/bacteriocin (Hly/Bac) production correlates with the presence of conjugative plasmids which transfer at a frequency of 10-1 to 10' in broth matings (23). A high percentage of the hemolysin plasmids bear structural similarities to the conjugative plasmid pAD1 (20,26). pAD1 is a 59.6-kilobase (kb) plasmid originally identified in E. faecalis DS16 (30) and belongs to the incompatibility class IncHly (9). The pAD1 hlylbac determinant was shown to contribute to pathogenicity in a mouse model (24). In addition to Hly/Bac synthesis, pAD1 has been shown to confer resistance to UV light (7) and a conjugative mating response to the peptide sex pheromone cAD1 secreted by recipient cells (5,11,13).Early studies on the physical and chemical nature of the E. faecalis Hly/Bac found the hemolytic activity to be heat sensitive and inhibited by a teichoic acid produced by hemolytic strains (10). Granato and Jackson (16) 50 ,uM isopropyl-,-D-thiogalactopyranoside (Sigma) and 0.01% 5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside (Sigma) were included in the media. Unless otherwise indicated, the medium used for other aspects of this study was brain heart infusion (BHI, Difco). Solid media were prepared by the addition of 1.5% (wt/vol) agar (Difco

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)

“…The CylL L and CylL S proteins are modified posttranslation by CylL M (2), and the modified CylL L and CylL S proteins are secreted via CylL B , which is the ATP-binding exporter (16). The extracellular cytolysin precursors CylL L and CylL S are converted to the active cytolysin by CylA (2,22). In an early study of the ␤-hemolysin/bacteriocin (cytolysin) determinant (22), two functional domains within the operon were identified and it was found that one region encodes the toxin precursor L component, which is now known to be encoded byCylL 1 , CylL 2 , CylM, and CylB, and the other region encodes an activator A component, which is now known to be encoded by CylA and CylI (2,8,9,17,18,39).…”

“…The extracellular cytolysin precursors CylL L and CylL S are converted to the active cytolysin by CylA (2,22). In an early study of the ␤-hemolysin/bacteriocin (cytolysin) determinant (22), two functional domains within the operon were identified and it was found that one region encodes the toxin precursor L component, which is now known to be encoded byCylL 1 , CylL 2 , CylM, and CylB, and the other region encodes an activator A component, which is now known to be encoded by CylA and CylI (2,8,9,17,18,39). In the complementation experiment between the A component-producing strain or the wild-type strain and the L component-producing strain on blood agar plates, the ␤-hemolysis zone occurred around or along the L component-producing strain (22), indicating that the A component activates the L component extracellularly and that the activated L component possesses the ␤-hemolysin/bacteriocin activity and an excess of extracellular A component is present in the culture medium of the wild-type strain (24).…”

“…The bacteriocin production assay was performed as described previously (22). The test for immunity to the bacteriocin was performed essentially as described previously (22).…”

“…The hemolysin/bacteriocin of pAD1 is associated with virulence in animal models (4,25,29), and this plasmid is considered to be a typical E. faecalis hemolysin/bacteriocin plasmid (21,31). The mechanism of hemolysin/bacteriocin production in E. faecalis has been studied in detail with the hemolysin/bacteriocin determinant on this plasmid (16,17,18,22,39). The active hemolysin/bacteriocin is produced by extracellular complementation of the two CylL factors (i.e., CylL L and cylL S ) and CylA.…”

“…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).…”