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transcriptional control of sex-pheromone-inducible genes on plasmid pad1 of enterococcus faecalis and sequence analysis of a third structural gene for (ppd1-encoded) aggregation substance.the expression of several neighbouring genes on plasmid pad1 that are necessary for conjugation depend on induction with sex pheromone cad1. analyses of transcripts by northern blot hybridization demonstrated that the genes sea1 (encoding surface exclusion protein) and asa1 (encoding aggregation substance) are transcribed independently. both genes are organized in different operons together with neighbouring open reading frames of unknown function. several transcripts could be identified for sea ...19921640831
the corynebacterium xerosis composite transposon tn5432 consists of two identical insertion sequences, designated is1249, flanking the erythromycin resistance gene ermcx.analysis of the 50-kb r-plasmid ptp10 from the clinical isolate corynebacterium xerosis m82b revealed that the erythromycin resistance gene, ermcx, is located on a 4524-bp composite transposable element, tn5432. the ends of tn5432 are identical, direct repeats of an insertion sequence, designated is1249, encoding a putative transposase of the is256 family. is1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. the nucleotide sequence of the 1754-bp tn5432 central region is 99 ...19958559800
pam401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from enterococcus faecalis.two novel enterococcus faecalis-escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of baca on the e. faecalis plasmid ppd1 were constructed. the vectors were named pmgs100 and pmgs101. pmgs100 was designed to overexpress cloned genes in e. coli and e. faecalis and encodes the baca promoter followed by a cloning site and stop codon. pmgs101 was designed for the overexpression and purification of a cloned protein fused to a strep-tag consisting of 9 amino acids at ...200111229919
enterococcus faecalis plasmid pad1-encoded fst toxin affects membrane permeability and alters cellular responses to lantibiotics.fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of enterococcus faecalis plasmid pad1. intracellular overproduction of fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. extracellular addition of synthetic fst had ...200312644486
antisense rna regulation by stable complex formation in the enterococcus faecalis plasmid pad1 par addiction system.the par stability determinant, encoded by the enterococcus faecalis plasmid pad1, is the only antisense rna regulated postsegregational killing system identified in gram-positive bacteria. because of the unique organization of the par locus, the par antisense rna, rna ii, binds to its target, rna i, at relatively small, interspersed regions of complementarity. the results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other ...200415375120
an intramolecular upstream helix ensures the stability of a toxin-encoding rna in enterococcus faecalis.the par stability determinant is required for the stable inheritance of the plasmid pad1 in its native host, enterococcus faecalis. it is the only antisense rna-regulated addiction module identified to date in gram-positive bacteria. it encodes two small, convergently transcribed rnas, rna i and rna ii. rna i encodes the fst toxin and rna ii acts as the antitoxin by interacting with rna i posttranscriptionally. as the toxin-encoding component of the system, it is important that rna i is more sta ...200919103923
molecular and genetic analysis of a region of plasmid pcf10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in enterococcus faecalis.exposure of enterococcus faecalis cells carrying the tetracycline resistance plasmid pcf10 to the heptapeptide pheromone ccf10 results in an increase in conjugal transfer frequency by as much as 10(6)-fold. pheromone-induced donor cells also express at least two plasmid-encoded surface proteins, the 130-kda sec 10 protein, which is involved in surface exclusion, and the 150-kda asc10 protein, which has been associated with the formation of mating aggregates. previous subcloning and transposon mu ...19911938961
aminoglycoside-resistant streptococcus and enterococcus species isolated from bovine mammary secretions.a total of 117 isolates representing four streptococcus species and 20 isolates representing two enterococcus species from bovine mammary secretions were examined for resistance to streptomycin, kanamycin, and gentamicin. resistance to streptomycin (85.4%) was most prevalent, followed by kanamycin (19%) and gentamicin (2.2%). minimum inhibitory concentration of streptomycin for most organisms examined ranged from 16 to 250 micrograms/ml. for kanamycin, the minimum inhibitory concentration for mo ...19921578037
genetic analysis of the pad1 hemolysin/bacteriocin determinant in enterococcus faecalis: tn917 insertional mutagenesis and cloning.thirty-seven nonhemolytic/nonbacteriocinogenic mutations in enterococcus (streptococcus) faecalis plasmid pad1 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 ...19902152897
emerging plasmid-encoded antisense rna regulated systems.classic antisense rna research has focused on detailed examination of a few plasmid-encoded systems whilst more recent efforts have focused on chromosomally encoded small rnas. recent work on newly identified plasmid-encoded antisense rnas suggest that there is still much to learn from them about the versatility of regulatory rnas. the alpha-proteobacterial repabc plasmids produce an antisense rna that regulates the replication initiator independently of the partition proteins encoded in the sam ...200717376732
sequence and analysis of the replication region of the staphylococcus xylosus plasmid psx267.the region of the 29.5-kb plasmid psx267 from staphylococcus xylosus dsm 20267 that is required for autonomous replication in staphylococci was isolated on a 1.8-kb dna fragment. the sequence analysis of the fragment yielded two open reading frames, repa and orf2, encoding proteins of 37.2 and 13.2 kda, respectively. the deduced amino acid sequence of repa showed similarity to the replication initiator protein of plasmid pad1 from enterococcus faecalis, to two proteins of unknown function encode ...19968982076
identification of the cad1 sex pheromone precursor in enterococcus faecalis.the enterococcus faecalis virulence plasmid pad1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cad1, secreted by plasmid-free enterococci. the determinant for the pheromone in e. faecalis fa2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cad1 moiety. the lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. the n ...200211889094
identification, characterization, and nucleotide sequence of a region of enterococcus faecalis pheromone-responsive plasmid pad1 capable of autonomous replication.a 5-kbp region of pad1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. transposon inserts identifying essential replication, copy control, and stability functions were isolated. deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. the complete dna sequence of this approximately 3-kbp reg ...19938384618
sex pheromone plasmid pad1-encoded surface exclusion protein of enterococcus faecalis.during conjugative transfer of sex pheromone plasmids of enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. we report here the dna sequence of a 3.8 kb fragment of the sex pheromone plasmid pad1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (orf) upstream of sea1. surface exclusion protein sea1 was found to be highly homologous ...19921603060
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. ...19947961506
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 ...19947531349
c-terminal identification of ad74, a proteolytic product of enterococcus faecalis aggregation substance: application of liquid chromatography/mass spectrometry.sexual aggregation involved in conjugative transfer of enterococcus faecalis plasmid pad1 is enhanced by the sex pheromone cad1, which is excreted from recipient cells. a membrane-anchored 137 kda protein is a pad1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. an ad74 protein is a proteolytic product corresponding to the n-terminal half of asal. the c-terminal of ad74 was identified as lysine at position 510 (k-51 ...19921368124
regulation of the pad1 sex pheromone response of enterococcus faecalis by direct interaction between the cad1 peptide mating signal and the negatively regulating, dna-binding traa protein.the enterococcus faecalis conjugative plasmid pad1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cad1. the response involves two key plasmid-encoded regulatory proteins: trae1, which positively regulates all or most structural genes relating to conjugation, and traa, which binds dna and negatively regulates expression of trae1. in vitro studies that included development of a dna-associated protein-tag affinity chromatography technique showed that traa (37.9 kd ...19989600983
solution structure and membrane binding of the toxin fst of the par addiction module.the par toxin-antitoxin system is required for the stable inheritance of the plasmid pad1 in its native host enterococcus faecalis. it codes for the toxin fst and a small antisense rna which inhibits translation of toxin mrna, and it is the only known antisense regulated toxin-antitoxin system in gram-positive bacteria. this study presents the structure of the par toxin fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. the mode of membran ...201020677831
cloning and characterization of the uvr (ultraviolet resistance) gene on conjugative plasmid pad1 of enterococcus faecalis. 19958586249
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