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

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...

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.Aminoglycosides such as kanamycin, gentamicin, and neomycin bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block growth through interference with protein synthesis (25). These agents have been used for the treatment of a broad range of life-threatening infections due to both gram-positive and gram-negative bacteria in human and veterinary medicine (18, 37). However, bacteria have acquired various aminoglycoside resistance mechanisms, such as through the production of aminoglycoside-modifying enzymes (acetyltransferase, nucleotidyltransferase, and phosphotransferase), the reduction of antibiotic penetration on the outer membrane protein, the acquisition of reduced affinity by changing key nucleotides within the 16S rRNA, and augmented excretion by an efflux pump system (5,25,36,42).In 2003, a plasmid-mediated 16S rRNA methyltransferase, which confers a high level of resistance to various clinically important aminoglycosides, was reported to be involved as part of a novel aminoglycoside resistance mechanism in pathogenic gram-negative rods (16, 53). At present, five types of plasmidmediated 16S rRNA methyltransferase genes, rmtA, rmtB, rmtC, rmtD, and armA, have been found worldwide in members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. (4,7,10,11,17,20,34,48,(50)(51)(52). Also, these genes are mediated by bacterium-specific recombination systems, such as transposons, and are easily translocated to other DNA target sites (17,19,47,49).The 16S rRNA methyltransferases conferring aminoglycoside resistance are supposed to have evolved as a self-defense mechanism in aminoglycoside-producing actinomyce...

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bacI, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bacI ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bacI, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broadspectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.It is known that many Enterococcus faecalis strains produce bacteriocins (3, 6). 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. However, it has become evident that many of the E. faecalis bacteriocins studied to date have a somewhat broader spectrum of activity, affecting more distantly related species (3,19,36). Bacteriocins are thought to provide the producer strain with a selective advantage over other strains and are a factor in bacterial virulence. The E. faecalis 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 specific peptide sex pheromones secreted by potential recipients (6,7,12,13,26,45,46). The sex pheromone induces the formation of a mating aggregate (6, 7, 12).To date, three types of E. faecalis bacteriocins have been genetically and biochemically characterized. These...