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At least 613 records · Page 34Linked to original sources
PubMed1994

Characterization of the determinant (traB) encoding sex pheromone shutdown by the hemolysin/bacteriocin plasmid pAD1 in Enterococcus faecalis.

pAD1 is a hemolysin/bacteriocin plasmid originally identified in Enterococcus faecalis DS16. It encodes a mating response to a peptide sex pheromone, cAD1, secreted by recipient bacteria. Once pAD1 is acquired, production of the pheromone ceases--a trait related in part to a determinant designated traB. Here we report the nucleotide sequence of traB and the position of several transposon insertions resulting in the characteristic self-induction phenotype. The deduced product has a mass of 43.7 kDa with the C-terminal third consisting primarily of hydrophobic amino acid residues.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.

Base Sequence↗
PubMed1994

Nucleotide sequence of the 18-kb conjugative transposon Tn916 from Enterococcus faecalis.

Conjugative transposon Tn916 from Enterococcus faecalis DS16 encodes tetracycline resistance (Tet M) as well as determinants necessary for its own movement. Determination of the nucleotide sequence of Tn916 has been completed. The element is 18,032 bp in length and has an overall G+C content of 38.8%. Twenty-four potential open reading frames (ORFs) were identified based on sequence analysis. Similarities of the ORFs to other known determinants, which were revealed by database searches, are discussed.

Base Sequence↗
PubMed1995

Dosage of Tn916 circular intermediates in Enterococcus faecalis.

Upon excision, conjugative transposon Tn916 forms a nonreplicating circular intermediate (CI), which is essential both for transposition and conjugal transfer. In this work we developed an assay to quantify the circular forms of Tn916. By this method we defined a new measurable trait of Tn916-carrying strains: the "CI copy number." CI dosage was performed by nested PCR (with primers designed on Tn916 termini) in a limiting dilution assay, where Poisson statistics were used to calculate the number of PCR amplification targets from the proportion of the negative endpoints. The number of CI was normalized to the number of bacterial chromosomes. This method enabled us to study the relationship between CI copy number, presence of tetracycline (the resistance marker of Tn916) in the culture medium, and conjugation frequency. Three isogenic strains of Enterococcus faecalis OG1 with Tn916 inserted at different sites on the chromosome were investigated for CI content. CI copy number varied depending on the strain, ranging between 7.8 and 610 copies per 10(6) chromosomes. Growth in liquid media containing tetracycline provoked an important increase both in CI copy number and conjugation frequency. This effect was more marked in low-frequency donors. While cell-cell contact during filter mating did not produce an increase in CI copy number, Tn916 conjugation frequency was found to be dependent on CI copy number in donor cells. The dose-response curve showed a linear relationship with a slope of 0.74, for the entire range of conjugation frequencies tested (from 5.1 x 10(-8) to 2.8 x 10(-6) transconjugants per donor.

Bacterial Adhesion↗
PubMed1996

Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation.

The prgB gene of the Enterococcus faecalis pheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target plasmids were constructed containing a recently identified positive control region and a prgB::lacZ transcriptional fusion; expression of prgB in cells carrying these plasmids was thus verified by beta-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as indentified by deletion analysis, was a 6.9-kb region extending from the 5' end of a gene called prgN, through a previously identified gene, prgX. The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that the prgN, -Y, and -X genes are involved in negative control; prgW may also play a role in negative control, since it appeared to be required for expression of prgY. prgX, or a closely adjacent DNA sequence, acted in cis. The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon in E. faecalis.

Amino Acid Sequence↗
PubMed1996

A novel tetracycline-resistant determinant, tet(U), is encoded on the plasmid pKq10 in Enterococcus faecium.

Nine tetracycline (Tc)-resistant clinical isolates of Enterococcus faecium were screened for plasmid content using agarose gel electrophoresis. pKQ10, a 1.9-kb plasmid carrying a novel Tc resistance determinant, was isolated from one of the isolates. The nucleotide sequence of this plasmid revealed an open reading frame corresponding to an 11.8-kDa protein and containing 105 amino acid residues. There was some limited similarity between this protein and tet(M), tet(O), tet(Q), tet(S), tetB(P), and otr(A), which overlapped, but did not include, the consensus GTP-binding sequences. The low-level, Tc-resistant determinant of pKQ10, named tet(U), does not appear to correspond to any other known Tc resistance determinant.

Amino Acid Sequence↗
PubMed1996

Sequences found on staphylococcal beta-lactamase plasmids integrated into the chromosome of Enterococcus faecalis CH116.

We have previously reported the presence of the staphylococcal beta-lactamase gene in chromosomes of Enterococcus faecalis strains CH19 and CH116. CH116 also harbors a 26-kb mobile element, designated Tn5384, which confers resistance to erythromycin and gentamicin. Sequence analysis of the rightmost 9 kb of Tn5384 indicates that this element lies immediately upstream of the beta-lactamase determinant in E. faecalis CH116. This 9-kb region consists of sequences highly homologous to those previously described in staphylococcal beta-lactamase plasmids, including a beta-lactamase transposon indistinguishable from Tn552, an open reading frame encoding a deduced amino acid sequence 94% identical to a previously described potential staphylococcal invertase, an intact copy of staphylococcal insertion-like element IS257, and the major portion of the staphylococcal organomercurial lyase (merB) gene. These data are consistent with the hypothesis that several of the resistance genes encoded within the large transferable region of the CH116 chromosome were originally components of a staphylococcal beta-lactamase plasmid.

Base Sequence↗
PubMed1996

The traA gene of the Enterococcus faecalis conjugative plasmid pPD1 encodes a negative regulator for the pheromone response.

The Enterococcus faecalis conjugative plasmid pPD1 (59 kb) produces an aggregation substance in response to pheromone. The traA gene (962 bp) is a member of a gene cluster involved in the regulation of the pPD1 pheromone response. A chimeric E. faecalis suicide plasmid containing a 762-bp DNA fragment from the middle portion of the traA gene was constructed. The pPD1 traA was disrupted by integration of this chimeric plasmid via homologous recombination. The E. faecalis strain containing pPD1 with the disrupted traA exhibited dry colony morphology, constitutive clumping, and an ability to transfer at high frequencies in a short (10-min) mating period, indicating that the traA product of pPD1 is a negative regulator for the pheromone response.

Bacterial Proteins↗
PubMed1996

Analyses of traA, int-Tn, and xis-Tn mutations in the conjugative transposon Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 moves intercellularly via an excision/insertion mechanism that involves products of int-Tn and xis-Tn. Tn5-insertion mutations in these genes were found to be complemented in an Enterococcus faecalis host by specific coresident transposons harboring the corresponding wild-type allele. A determinant designated traA, partially overlapping and divergently transcribed from xis-Tn, is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express a trans-acting product.

Amino Acid Sequence↗
PubMed1997

The joint of Tn916 circular intermediates is a homoduplex in Enterococcus faecalis.

Tn916 is a 18-kb conjugative transposon originally identified in Enterococcus faecalis. The first step for Tn916 movement is its excision from the donor replicon with the formation of a nonreplicating covalently closed circular intermediate. Studies on formation of circular intermediates in Escherichia coli have shown that the joint between the Tn916 termini is a 6-bp heteroduplex formed by the two regions flanking the transposon before its excision (coupling sequences). In this work we studied the joint of Tn916 termini in circular intermediates formed in both E. coli and E. faecalis. Our strategy was to use direct sequencing of amplification products obtained from the joint region of single target molecules. In E. coli, 50% of circular intermediates contained a heteroduplex joint, while the remaining 50% displayed a homoduplex joint formed by one of the two coupling sequences. In E. faecalis, we could not demonstrate the presence of any heteroduplex joint. In this case 77.7% of the analyzed joints were formed by the left coupling sequence.

DNA Replication↗
PubMed1998

Isolation of a derivative of Escherichia coli-Enterococcus faecalis shuttle vector pAM401 temperature sensitive for maintenance in E. faecalis and its use in evaluating the mechanism of pAD1 par-dependent plasmid stabilization.

A derivative of the Escherichia coli-Enterococcus faecalis shuttle vector pAM401 was isolated by mutagenesis in an E. coli mutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38 degreesC than at 30 degreesC in the E. faecalis host strain JH2-2. The E. faecalis plasmid pAD1-encoded par stability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found that par stabilized pAM401ts at 38 degreesC but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38 degreesC. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putative par antidote, in trans attenuated cell killing. These characteristics support a postsegregational killing mechanism for par-mediated plasmid stabilization.

DNA Replication↗
PubMed1999

Enterococcus faecalis pheromone-responding plasmid pAD1 gives rise to an aggregation (clumping) response when cells are exposed to subinhibitory concentrations of chloramphenicol, erythromycin, or tetracycline.

The Enterococcus faecalis conjugative cytolysin plasmid pAD1 encodes a specific aggregation (clumping) response to the peptide sex pheromone cAD1 secreted by plasmid-free strains. Here it is shown that, in the absence of cAD1, exposure of E. faecalis cells harboring pAD1 to subinhibitory concentrations of chloramphenicol, erythromycin, or tetracycline also results in an aggregation response that appears related to a stress-sensitive mechanism associated with a component of the pheromone response.

Anti-Bacterial Agents↗
PubMed1999

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.

Amino Acid Sequence↗
PubMed2000

Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing.

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 has been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcus faecalis to serve as a donor for the conjugative transfer of transposon Tn916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of > or =6 x 10(-8) per recipient CFU. To determine the transposon insertion site, an oligonucleotide primer corresponding to the 3' end of Tn916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown to insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepticum genome. The ability to determine transposon insertion sites in mycoplasmas by genomic sequencing has not been previously described and allows rapid sequence analysis of transposon-generated mutants.

Animals↗
PubMed2001

Completion of the nucleotide sequence of the Enterococcus faecalis conjugative virulence plasmid pAD1 and identification of a second transfer origin.

pAD1 is a 59.3-kb plasmid in Enterococcus faecalis that has been the subject of intense investigation with regard to its pheromone-inducible conjugation behavior as well as its contribution to virulence. Approximately two-thirds of the pAD1 nucleotide sequence has been previously reported. Here we report on an analysis of the final approximately 22 kb, a significant portion of which is believed to encode structural genes associated with conjugation. The conjugation-related region was also found to contain a new (second) origin of conjugative transfer (oriT). A list of open reading frames covering the entire plasmid is presented.

Conjugation, Genetic↗
PubMed2001

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.

Animals↗
PubMed1990

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775.

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.

Acetyltransferases↗