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D B Clewell

Publications and source records attributed to D B Clewell.

At least 19 recordsLinked to original sources
PubMed1995

Targeted disruption of the PD78 gene (traF) reduces pheromone-inducible conjugal transfer of the bacteriocin plasmid pPD1 in Enterococcus faecalis.

Bacterial sex pheromone, cPD1, induces sexual aggregation of Enterococcus faecalis harboring the bacteriocin plasmid, pPD1, and enables pPD1 to transfer at high frequency in a liquid culture. PD78 is a cPD1-inducible cell surface protein encoded by pPD1. The PD78 gene, traF, was disrupted by homologous recombination between pPD1 and an artificial vector having a deletion in the middle portion of traF. The disruption of traF did not affect the cPD1-inducible aggregation but reduced the transfer frequency of pPD1 to 2% of the wild-type level.

Bacterial Proteins↗
PubMed1995

Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons.

Conjugative transposons are highly ubiquitous elements found throughout the bacterial world. Members of the Tn916-Tn1545 family carry the widely disseminated tetracycline-resistance determinant Tet M, as well as additional resistance genes. They have been found naturally in, or been introduced into, over 50 different species and 24 genera of bacteria. Recent investigations have led to insights into the molecular basis of movement of these interesting mobile elements.

Bacteria↗
PubMed1995

Oral streptococci with genetic determinants similar to the glucosyltransferase regulatory gene, rgg.

The Streptococcus gordonii Challis glucosyltransferase structural gene, gtfG, is positively regulated by the upstream gene, rgg, the only described gtf regulatory determinant in oral streptococci. Southern hybridization analyses indicated that rgg-like and gtfG-like determinants were present on the same HindIII fragment in strains of S. gordonii, Streptococcus sanguis, and Streptococcus oralis, whereas no rgg-like determinants were detected in mutans streptococci, Streptococcus mitis, and Streptococcus salivarius.

Base Sequence↗
PubMed1995

Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region.

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.

Bacterial Proteins↗
PubMed1995

Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes.

Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown.

Amino Acid Sequence↗
PubMed1995

A functional origin of transfer (oriT) on the conjugative transposon Tn916.

The origin of transfer (oriT) of the 18-kb conjugative transposon Tn916 has been localized to a 466-bp region which spans nucleotides 15215 to 15681 on the transposon map. The oriT lies within an intercistronic region between open reading frames ORF20 and ORF21 that contains six sets of inverted repeats ranging from 10 to 20 bp in size. The segment contains three sequences showing identity in 9 of 12 bp to the consensus nicking site (nic) of the IncP family of conjugative plasmids found in gram-negative bacteria. Overlapping one of these sequences is a region similar to the nic site of the F plasmid. Functionality was based on the ability of the oriT-containing sequence to provide a cis-acting mobilization of chimeras involving the shuttle vector pWM401 in response to activation in trans by an intact chromosome-borne transposon Tn916 delta E. Cloned segments of 466 or 376 nucleotides resulted in unselected cotransfer of the plasmid at levels of about 40% when selection was for Tn916 delta E, whereas a 110-bp segment resulted in cotransfer at a frequency of about 7%. Mobilization was specific in that gram-positive plasmids, such as pAD1 and pAM beta 1, and the gram-negative plasmids pOX38 (a derivative of F) and RP1 did not mobilize oriT-containing chimeras.

Base Sequence↗
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

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↗
PubMed1994

Tn924, a chromosome-borne transposon encoding high-level gentamicin resistance in Enterococcus faecalis.

Enterococcus faecalis SF350 is a clinical isolate from Winnipeg, Canada, with high-level (MIC > 2,000 micrograms/ml) gentamicin resistance. The genetic determinant for gentamicin resistance was located on the chromosome of SF350 and could be mobilized by a coresident conjugative plasmid, pYN120. Genetic and physical analyses showed that the gentamicin resistance determinant was located on a 27-kb transposable element which was designated Tn924.

Chromosomes, Bacterial↗
PubMed1994

Evidence that coupling sequences play a frequency-determining role in conjugative transposition of Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 (encodes resistance to tetracycline), originally identified in Enterococcus faecalis, moves by an excision-insertion process in which the rate-limiting step is believed to be excision. Individual transposon-containing strains exhibit characteristic mating frequencies which range over several orders of magnitude; the basis of this phenomenon is addressed in the present study. We were able to generate independent single-copy insertions in identical target locations and with similar orientations within a plasmid hemolysin determinant (cylA); however, transposition from this site occurred at very different frequencies (10(-8) to 10(-4) per donor) depending on the individual isolate. DNA sequencing analyses showed that the coupling (junction) sequences differed between isolates and thus appeared to be responsible for differences in excision frequencies. Other experiments showed that inducible transcription into either end of the transposon had no significant effect on transfer.

Base Sequence↗
PubMed1994

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.

Adenosine Triphosphate↗
PubMed1993

Infectivity of a glucan synthesis-defective mutant of Streptococcus gordonii (Challis) in a rat endocarditis model.

Streptococcus gordonii, a member of the human indigenous oral microflora, colonizes smooth tooth surfaces and contributes to dental plaque formation. Although it is not recognized as being a cariogenic pathogen, it may cause endocarditis following invasion of the bloodstream. Using allelic exchange mutagenesis, we have constructed a mutant of S. gordonii (Challis) which is defective in its single functional glucosyltransferase gene and, hence, is unable to synthesize glucan exopolymers from sucrose. When examined in a rat endocarditis model, the sucrose-grown mutant did not differ significantly from S. gordonii wild-type, suggesting that glucan polymers did not contribute to infectivity. This result was in striking contrast to that previously observed with a polymer-defective S. mutans mutant.

Animals↗
PubMed1993

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.

DNA Restriction Enzymes↗
PubMed1993

Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis.

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

Animals↗
PubMed1993

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.

Amino Acid Sequence↗