Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1

Abstract

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...

Effect of Bacteriocin-like Substance Produced by Enterococcus faecium EF55 on the Composition of Avian Gastrointestinal Microflora

“…This could be due to secondary interactions between intestinal microflora groups. Moreover, activity of enterocins towards Gram-negative bacteria like E. coli (Tomita et al 1997) and Vibrio cholerae has been shown (Simonetta et al 1997).…”

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

“…Subsequent searches of the NR database using PSI-BLAST showed that homologues of g2635842 occur in Streptococcus crista (TptB), Enterococcus faecalis (BacG), Lactobacillus sake (SapE), and another in B. subtilis (g2633806). The three named proteins are putative components of ABC transport complexes (Axelsson & Holck, 1995;Correia et al, 1997;Tomita et al, 1997). All four of these proteins also have clear ®rst lipoyl motifs (M N ) and are immediately followed by regions with a high probability (>0.9) of forming coiled coils.…”

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)

“…Mutant pHT1100 plasmids with BamHI fragment deletions within were also generated to examine the location of the bacteriocin determinant as described in Materials and Methods (Fig. 2) (47). Deletion mutant plasmids pMG1103 and pMG1104 possessed deletions of the 4.1-kbp BamHI E fragment between map positions 0 kb and 4.1 kb and the 2.2-kbp BamHI F fragment between map positions 4.1 kb and 6.3 kb, respectively.…”

“…Insertion of Tn5 (Km r ) into the cloned plasmid DNA was performed as described elsewhere (47). Target plasmid pHT1100(pAM401 containing EcoRI fragments A and H) was introduced into E. coli K-12 TH688 (with Tn5 in the thr locus) (42) by electrotransformation.…”

“…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).…”