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...
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...
The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Em r ) or Tn916 (Tc r ) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF-orfY-sea-1 with all but repB and traA oriented in the same (left-toright) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.Certain conjugative plasmids in Enterococcus faecalis transfer at a high frequency in broth mating, a phenomenon relating to a response to specific peptide sex pheromones secreted by potential recipients. The sex pheromone induces the synthesis of a surface aggregation substance that facilitates the formation of a mating aggregate (5-7, 14, 15, 18, 48). When a given plasmid is acquired, secretion of the related pheromone is prevented; however, unrelated pheromones continue to be produced. In addition, the plasmid itself determines the production of a peptide that acts as a competitive inhibitor of the corresponding pheromone (8,10,11,27,32).Several pheromone-responding plasmids have been reported, and the plasmids in E. faecalis have been found to encode such traits as hemolysin-bacteriocin production, UV resistance, and drug resistance (5-7). Of these plasmids, the pheromone-related conjugation systems best studied are pAD1 (60 kb) (6,7,9,12,23,33,35,36,40,41,44,45) and pCF10 (54 kb) (4, 30, 37), which confer responses to sex pheromones cAD1 and cCF10, respectively. pAD1 encodes a hemolysin-bacteriocin mediated by the same genetic determinant (2, 3, 5, 26) and belongs to incompatibility group incHly (13,25). Most (over 90%) of the Hly-Bac plasmids identified in clinical isolates are identical and exhibit extensive homology to pAD1, respond to cAD1, and represent incompatibility group incHly (24,25,29). pAD1 is a representative of this group. pCF10 carries a tetracycline resistance determinant located on transposon Tn925. Genes involved in regulation of the pheromone response have been identified and are clustered in a 7-kb region on each of the plasmids. In pAD1, these genes are arranged in the order traE1-iad-traA-traC-traB (6). The traE1 product is a key positive regulator for expression of downstream structural genes, including determinant asa-1 for the aggregation substance and other conjugation-related genes.The traA product has been shown to represent a key negative regulator of traE1 expression. The traB product is involved in shutdown of endogenous cAD1 p...
The potencies of the carbapenems; doripenem (DRPM), meropenem (MEPM) and imipenem (IPM) in preventing the emergence of carbapenem-resistant mutants were examined in Pseudomonas aeruginosa strains. The carbapenems predominantly selected carbapenem-resistant mutants or carbapenem mutants with reduced susceptibilities that were specifically resistant to carbapenems and had arisen as a result of the reduced level of expression of the outer membrane protein with a molecular weight of about 48,000 (OprD). The potency of carbapenems in preventing the growth of the mutants differed for DRPM, MEPM and IPM. The isolation frequency of the mutant was examined on agar plates containing each of the carbapenems at a concentration of 1/2 or 1/4 MIC of each carbapenem for that mutant. Mutants were not selected on agar containing DRPM at a frequency of greater than 10 Ϫ9 per cell per generation, whereas mutants of each strain were selected on agar containing MEPM or IPM at frequencies of 10 Ϫ7 to 10 Ϫ9 per cell per generation. The drug concentrations and the drug concentration range for the selective increase of carbapenem resistant mutants in the broth culture containing each carbapenem differed for each carbapenem. DRPM exhibited both the lowest drug concentration and the narrowest range of drug concentration for selection of the carbapenem-resistant mutants. The results shown in this report indicated that DRPM exhibited the greatest ability to prevent the emergence of the mutant.
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