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Results for “plasmid characterization”

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PubMed1983

Plasmid-determined antibiotic resistance in Shigella flexneri isolated in England and Wales between 1974 and 1978.

The majority of Shigella flexneri strains isolated in England and Wales are from infections contracted abroad. Most of these strains are drug-resistant, over 75% being resistant to streptomycin and sulphonamides, sulphonamides alone or streptomycin, sulphonamides and tetracyclines. A selection of resistant strains was tested for resistance transfer and the plasmids identified were characterized by compatibility grouping. Streptomycin and sulphonamide resistance was usually determined by a non-autotransferring plasmid which may be mobilized by standard transfer factors, or by the plasmid which conferred tetracycline resistance where this was present. The remaining resistant strains were predominantly resistant to four or more drugs. These strains carried autotransferring plasmids of a variety of compatibility groups, of which groups B, I1 and FII were the most common.

Ampicillin↗

PubMed2004

Construction and characterization of a highly efficient Francisella shuttle plasmid.

Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 x 10(7) CFU mug of DNA(-1) in F. tularensis LVS, Francisella novicida U112, and E. coli DH5alpha. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

Anti-Bacterial Agents↗

PubMed2000

Cloning and characterization of the replicon of the Nocardia italica plasmid, pNI100.

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.

Amino Acid Sequence↗

PubMed1997

Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ.

Shigella species and enteroinvasive Escherichia coli contain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains of Shigellae. Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen from Shigella flexneri, designated IpaJ. The ipaJ gene encodes a 780 bp open reading frame (ORF), separated from the ipaR (virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoA mutagenesis of the ipaJ ORF confirmed the presence of export signal sequences in IpaJ. Unlike ipaBCDA genes, transcription analysis of ipaJ indicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as a Shigella immunogen. Construction and phenotypic characterization of ipaJ mutants in two serotypes of S. flexneri showed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, the ipaJ mutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.

Amino Acid Sequence↗

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↗

PubMed1999

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus.

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators, pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS257 was not involved in the recombination events which gave rise to pRJ29.

Bacteriocins↗

PubMed1996

Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.

Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.

Animals↗

PubMed1984

DNA sequences from a ligninolytic filamentous fungus Phanerochaete chrysosporium capable of autonomous replication in yeast.

Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated. These hybrid plasmids with the autonomous replication sequences from P. chrysosporium are maintained extra-chromosomally, are mitotically unstable and transform Ura3 deletion mutant of S. cerevisiae to Ura+ phenotype with high frequency. The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P. chrysosporium genomic DNA. Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning.

Base Sequence↗

PubMed1995

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060. The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly described palT type SSOs. Also pTA1020 and pTA1060 were shown to contain SSOs highly homologous to palT. Using Southern hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned. Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA1015. The plasmids pTA1030 and pTA1050 contain an SSO which is highly homologous to the SSO of pTA1040. The majority of the SSOs of rolling-circle plasmids from B.subtilis seem to belong to two related families which we denote as palT1 (present on pTA1015, pTA1020 and pTA1060) and palT2 (present on pTA1030, pTA1040 and pTA1050). Both families of SSOs are highly efficient single-strand-conversion signals in B.subtilis.

Bacillus subtilis↗

PubMed1999

Molecular typing of multiresistant Klebsiella pneumoniae isolated from children from northern Jordan.

Twenty-nine clinical isolates of community acquired Klebsiella pneumoniae obtained from 17 children with malnutrition were characterized by antibiotic susceptibility, plasmid analysis, and random amplified polymorphic DNA (RAPD) techniques. Disc diffusion methodology was used to test the susceptibility of the isolates to 13 antibiotics: amoxycillin, cephapirin, ceftazidime, cefoxitin, cefotaxime, aztreonam, gentamicin, ciprofloxacin, chloramphenicol, erythromycin, nalidixic acid, trimethoprim and amoxycillin-clavulanic acid. All the isolates showed multiresistance patterns (15 patterns) ranging from resistance to two antibiotics to resistance to 10 antibiotics. All isolates were resistant to amoxycillin and erythromycin. Ten K. pneumoniae isolates producing extended-spectrum beta-lactamases (ESBLs) as evidenced by the double-disc diffusion synergy test were isolated sporadically from six patients. Six of these 10 isolates were hyperproducers of ESBL, which resulted in increased resistance to the beta-lactam-beta-lactamase inhibitor combination amoxycillin-clavulanic acid. Plasmid analysis showed plasmid ranging in size from 48 kilobases (kb) to 1.4 kb. All the 29 isolates shared the same plasmid 26 kb. There was a consistent relationship between antibiotype and plasmid profiles for each pair of isolates obtained from five individual patients. RAPD analysis using a single (10-mer) primer demonstrated that the isolates that have the same antibiotype and the same plasmid profile had different RAPD fingerprint patterns. These results demonstrate that the RAPD technique is better than antibiotype characterization and a plasmid analysis profile for typing K. pneumoniae as well as for revealing strain differences.

Anti-Bacterial Agents↗

PubMed1982

Novel antibiotic resistance transfer in Bacteroides.

Resistance to tetracycline and lincosamide antibiotics was transferred en bloc from a strain of Bacteroides fragilis (V503) to a plasmidless strain of Bacteroides uniformis (V528) during in vitro filter matings. Resistance transfer was detected at frequencies of 10(-5) to 10(-6) drug-resistant progeny per input donor cell and was dependent on cell-to-cell contact of donors and recipients. Transfer was insensitive to DNase and was not mediated by chloroform- or filter-sterilized donor broth cultures. A determinant for resistance to cefoxitin in V503 was not transferred in this system. V503 contained a 3.7 x 10(6)-dalton plasmid (pVA503). Drug-resistant progeny of V503 x V528 matings usually contained pVA503, but up to 20% of the total progeny of such crosses were plasmid free. Filter blot DNA hybridization studies (Southern method) confirmed that pVA503 was not integrated into the host chromosome of the plasmidless progeny. Drug-resistant progeny from V503 x V528 matings (with or without pVA503) conjugally transferred clindamycin resistance an tetracycline resistance to a suitable recipient strain. None of the drug resistance determinants of V503 were affected by treatment with standard plasmid curing regimens, and methods designed to detect very large plasmid molecules failed to suggest the involvement of extrachromosomal DNA in this resistance transfer system. The well-characterized Bacteroides R plasmid, pBF4 (conferring clindamycin resistance), was found to share hybridizing sequences with bulk cellular V503 DNA when examined by filter blot hybridization. Similarly sized sequences were found in drug-resistant progeny recovered from matings. Neither of the two pBF4 derivatives carrying deletions that abolished clindamycin resistance hybridized with V503 DNA.

Anti-Bacterial Agents↗

PubMed1989

Characteristics and restriction analysis of the 4-chlorobiphenyl catabolic plasmid, pSS50.

The plasmid pSS50 is a 53-kilobase self-transmissible plasmid of broad host range that has been isolated from several Alcaligenes and Acinetobacter species. This plasmid has previously been shown to mediate the mineralization of 4-chlorobiphenyl to carbon dioxide and water. Physical characterization of this plasmid by restriction analysis indicates that most hexanucleotide cleavage sites are clustered in a 5-kilobase region, leaving large regions without restriction sites. The paucity of restriction sites is not due to DNA methylation.

Acinetobacter↗

PubMed1978

Bacteriophage mu-induced deletions in a plasmid containing the nif (N2 fixation) genes of Klebsiella pneumoniae.

Five plasmids with insertions of a heat-inducible Mu prophage in a Mu-sensitive and P1-sensitive derivative of plasmid pRD1, a recombinant R factor containing the his-nif region of Klebsiella pneumoniae, were isolated and characterized. In one plasmid containing the Mu prophage integrated at the his-distal end of nif, selection for heat resistance resulted in the generation of deletions extending from the Mu prophage into the nif region. Thirty of these deltions were used to map 26 point mutations in nif.

Bacteriophages↗

PubMed1986

Identification of pheromone-induced surface proteins in Streptococcus faecalis and evidence of a role for lipoteichoic acid in formation of mating aggregates.

The conjugative transfer of the Streptococcus faecalis plasmid pAD1 is characterized by a 10,000-fold increase in frequency following sex pheromone (cAD1) induction. Before the increase in plasmid transfer, donor cells synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. Four novel surface proteins appearing after exposure of pAD1-containing cells to sex pheromone have been identified. Thirty minutes after induction, a 130-kilodalton (kDa) protein was detectable by Western blotting. A 74-kDa protein, the major species present, and a pair of bands at 153 and 157 kDa were evident 45 min after induction. Induced cells containing another conjugative S. faecalis plasmid, pPD1, gave rise to three high-molecular-weight proteins of the same size (130, 153, and 157 kDa) as those synthesized by pAD1-containing cells. These proteins cross-reacted with antisera raised against induced cells containing pAD1. However, the major protein species produced by pPD1-containing cells had a molecular weight of 78,000 and did not cross-react significantly with the corresponding band of the pAD1 system. Pheromone-induced transfer of the two plasmids, when both were present in the same cell, was independent; induction was limited to the pheromone-specified plasmid. The possibility that lipoteichoic acid might act as a receptor (binding substance) for the induced adhesin protein was also explored. Free lipoteichoic acid (isolated from S. faecalis) inhibited clumping of induced cells, apparently by acting as a competitive inhibitor of the cellular binding substance.

Bacterial Proteins↗

PubMed1994

Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization.

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.

Agrobacterium tumefaciens↗

PubMed1999

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization.

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol). Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short. We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence.

Artifacts↗

PubMed1979

Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids.

A modified in vitro replication system has been characterized and used to catalogue the host proteins required for the replication of plasmid RSF1030. These extracts differ from systems described previously in that endogenous DNA is removed. Replication in vitro therefore requires an exogenouos RSF1030. Synthesis in the in vitro system faithfully mimics in vivo replication with respect to the products synthesized, effects of specific inhibitors, and requirements for RNA polymerase and DNA polymerase I. In addition, we find that proteins encoded by dnaB, dnaC, dnaG, dnaI, dnaP and polC (DNA polymerase III), are required for in vitro plasmid synthesis. The product of dnaA is not required. Extracts prepared from E. coli mutants deficient in in vitro replication can be complemented by addition of purified proteins or of extracts carrying the wild type protein.

DNA Replication↗

PubMed1985

Relatedness of tetracycline resistance plasmids among species of coagulase-negative staphylococci.

Four isolates of Staphylococcus aureus and 98 isolates of coagulase-negative staphylococci representing six species all obtained from endocervical cultures were examined for antimicrobial susceptibility and for the presence of plasmids. More than 80% of the isolates were susceptible to each of 12 antimicrobial agents tested, whereas only 33% were susceptible to penicillin G, 30% were susceptible to cadmium chloride, and 41% were susceptible to tetracycline. Although no species-related susceptibility or plasmid patterns were detected, 77 isolates contained at least one plasmid and 43 contained a plasmid similar in mass to a 2.7-megadalton tetracycline resistance plasmid previously reported in staphylococci. Association of tetracycline resistance with plasmids of this size in four species was determined from curing experiments. No plasmids homologous with the tetracycline resistance locus of the Escherichia coli plasmid pBR322 were found among 11 isolates examined by DNA hybridization. Homology with a 2.7-megadalton plasmids (pRC701) from an endocervical isolate of S. aureus, however, was apparent for 2.7-megadalton plasmids harbored by six isolates as well as with larger plasmids harbored by three isolates. Restriction analysis revealed that pRC701 shared structural identity with two plasmids of a similar mass from two species of coagulase-negative staphylococci as well as with a previously characterized tetracycline resistance plasmid originating in S. aureus.

Base Sequence↗