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Results for “plasmid characterization”

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PubMed1988

[Isolation and characterization of gentamicin resistance plasmids of coliform bacteria from the waste water of a water treatment plant].

Gentamicin resistance coliforms detected continuously in raw and purified waste water samples of a sewage treatment plant made up less than 0.1% of all coliforms. 43.9%; 31.4%; 13.3% and 11.3% of gentamicin-resistant coliforms were identified as Enterobacter, E. coli, Klebsiella and Citrobacter, respectively. R plasmids encoding a gentamicin resistance phenotype were isolated and characterized. They range between 55 and 60 MD in size and belong to 3 incompatibility groups (IncOF, IncM, IncK). Using restriction endonucleolytic digestion of plasmids, they could be further characterized and subtyped. In contrast to moderate molecular alterations observed among the IncM plasmids, the IncOF plasmids reveal a high stability of restriction pattern. This IncOF plasmid was predominantly found in E. coli wild strains and detected continuously.

Citrobacter↗

PubMed1979

Physical characterization of ten R plasmids obtained from an outbreak of nosocomial Klebsiella pneumoniae infections.

Gentamicin resistance in Klebsiella pneumoniae involved in an outbreak at the Minneapolis Veterans Administration Hospital was due to a transmissible R plasmid. In addition to gentamicin, this plasmid conferred resistance to tobramycin, kanamycin, ampicillin, carbenicillin, cephalothin, chloramphenicol, and sulfathiazole. R plasmids which transferred this complex antibiogram were identified in several clinical isolates, including four different serotypes of K. pneumoniae, Escherichia coli, Enterobacter cloacae, and Proteus morganii. The covalently closed circular form of all R plasmids isolated had a sedimentation coefficient of 76S to 77S, corresponding to a molecular weight of 58 x 10(6). The possibility that a single R plasmid was responsible for the dissemination of multiple drug resistance among all of these different clinical strains was examined by characterizing the plasmids by using EcoRI restriction endonuclease. The same 15 fragments were obtained from each of the 10 plasmids analyzed. Their molecular weights ranged from 4 x 10(5) to 11 x 10(6). Thus, we conclude that each of the 10 plasmids present in the various clinical strains isolated from the hospital over a 7-month period originated from a common source and that R plasmid transfer was important in their spread.

Anti-Bacterial Agents↗

PubMed2003

Characterization of the double-partitioning modules of R27: correlating plasmid stability with plasmid localization.

Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form. Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1(-) and Par2(-)) under different growth conditions in live Escherichia coli cells. Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells. Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E. coli. Both R27 Par(-) variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm. Those cells that did contain R27 Par(-) with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27. Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm.

Adenosine Triphosphatases↗

PubMed1979

Incidence of antibiotic-resistant Escherichia coli associated with frozen chicken carcasses and characterization of conjugative R plasmids derived from such strains.

Escherichia coli were isolated at concentrations of about 10(2)/ml from the fluid obtained after thawing each of five frozen chicken carcasses. Between 13 and 89% of the E. coli were resistant to mercury(II) or to at least one of eight antibiotics tested. Multiple resistance was more common than single resistance, and resistances to tetracycline, streptomycin, sulfathiazole, or chloramphenicol were more frequently encountered than was resistance to ampicillin or mercury(II). Resistance to kanamycin, gentamicin, or trimethoprim occurred rarely. Upwards of 30% of the E. coli apparently carried conjugative plasmids and could transfer at least one of their resistance determinants to E. coli K-12. Twenty-five conjugative R plasmids obtained in this way were characterized within strain K-12 by a variety of phenotypic criteria. Twenty could be assigned to the incompatibility groups Inc X, Inc N, Inc FI, or Inc I alpha.

Animals↗

PubMed2001

Construction and initial characterization of an infectious plasmid clone of a newly identified hamster parvovirus.

The construction and characterization of a full-length infectious plasmid clone of the newly identified hamster parvovirus (HaPV) are described. Following transfection of hamster BHK cells with the infectious clone, pHaPV, the specific intracellular DNA replicative forms, RNA transcripts and viral proteins that were expected for this rodent parvovirus were generated. Infected cells were lysed and progeny virus was produced, demonstrating that pHaPV could generate a productive virus infection. The complete sequences of both hairpin termini, which had not been previously determined, were obtained. Preliminary host-range studies, which compared virus production and macromolecular synthesis in various cell lines following either HaPV infection or pHaPV transfection, demonstrated an early block of infection of HaPV in both monkey COS-1 and murine A9 cells. The availability of an HaPV infectious clone will facilitate its genetic analysis and allow the elucidation of the determinants important in host range, tissue tropism and pathogenicity of this newly identified rodent parvovirus.

Animals↗

PubMed1993

Characterization of the small cryptic plasmid, pIMVS1, of Salmonella enterica ser. Typhimurium.

The nucleotide sequence has been determined of a small cryptic plasmid of Salmonella Typhimurium, designated pIMVS1, which correlated with an outbreak of gastroenteritis. This plasmid has a size of 3357 bp with no known functions. It does not encode any protein products but shows homology to several well-characterized plasmids. The replication system with RNAI and RNAII and the replication origin (oriV) is highly similar to that of plasmid p15A. It also has a putative mobilisation origin similar to that of ColE1.

Base Sequence↗

PubMed1992

Characterization of two circular plasmids from the marine diatom Cylindrotheca fusiformis: plasmids hybridize to chloroplast and nuclear DNA.

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.

Cell Nucleus↗

PubMed1979

Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.

Anti-Bacterial Agents↗

PubMed1981

Cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives suggest molecular mechanisms for P1-mediated transduction of small plasmids.

We characterized cointegrates formed in an Escherichia coli rec+ strain between bacteriophage P1 genomes and small plasmids related to pBR322. The partners were, on the one hand, either phage P1 DNA, which carries one copy of IS1, or phage P1-15 DNA, a derivative which lacks the IS1, and, on the other hand, plasmids containing either a split IS1 or no. In the presence of IS1 sequences on both partners, cointegrates were usually formed by reciprocal recombination between SI1 sequences. Cointegrates between P1 and a plasmid carrying no IS1 sequence were formed by transpositional cointegration mediated by IS1 of P1. Cointegrates between P1-15 and small plasmid containing a split IS1 were formed by one of three ways: (a) acquisition of an IS1 by P1-15 followed by reciprocal recombination between IS1 sequences, (b) transpositional cointegration mediated by the split IS1 element, Tn2657, or (c) involvement of the invertible segment carried on P1-15 DNA. Most cointegrates segregated into the small plasmids and phage P1 derivatives. A comparison of the phenomenon studied and of their frequencies allowed us to conclude that cointegrate formation is a molecular mechanism involved in the transduction of plasmids smaller than those packageable into P1 virions, although it does not seem to be the only process used.

Bacteriophages↗

PubMedDate not supplied

Frequency in the United States of non-beta-lactamase-producing strains of Neisseria gonorrhoeae possessing the 24.5-Mdal conjugative plasmid.

We characterized 475 non-beta-lactamase-producing gonococcal isolates from five U.S. cities by auxotype, serovar, and plasmid content to determine the frequency of isolates with the 24.5-Mdal gonococcal conjugative plasmid. A total of 150 (32%) of all isolates possessed the 24.5-Mdal plasmid; these isolates represented between 10% and 53% of isolates from each city. Isolates possessing the 24.5-Mdal plasmid belonged to 45 (56%) of 81 auxotype/serovar (A/S) classes, or between 24% and 70% of the A/S classes isolated in each city. Overall, a higher proportion of strains isolated in the southern cities possessed the 24.5-Mdal plasmid than did those from northeastern or northwestern cities. These strains appear to have spread independently of the spread of beta-lactamase-producing Neisseria gonorrhoeae strains. The prevalence of N. gonorrhoeae strains with the 24.5-Mdal conjugative plasmid has increased substantially and may have important implications for the epidemiology of gonorrhea caused by beta-lactamase-producing strains.

Conjugation, Genetic↗

PubMed1989

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.

Adenine Nucleotides↗

PubMed1993

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.

Anthracenes↗

PubMed1985

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.

Bacteroides↗

PubMed1981

Characterization of the Streptococcus mutans plasmid pva318 cloned into Escherichia coli.

We further characterized the cryptic plasmid pVA318 of Streptococcus mutans. It had a contour length of 5.64 +/- 0.26 kilobases and a guanine-plus-cytosine content of 32 to 34 mol %. Upon cloning the pVA318 plasmid into the vector pBR322 in Escherichia coli, we made the following observations. The expression of tetracycline resistance by HindIII-cloned chimeras, where the insert was in the tetracycline resistance promotor, depended on the orientation of the pVA318 insert. Both HindIII-cloned chimeras segregated from polA(Ts) cells at a nonpermissive temperature. Chimeric molecules cloned with PstI initially showed much instability; the reason for this is unknown, although stable variants were obtained. Both HindIII-cloned variants and a PstI-cloned chimera produced a pVA318-specific protein of approximately 20,000 molecular weight in E. coli minicells. The biological function of this protein is not known; it had no bacteriocin activity against S. mutans or group A Streptococcus indicator strains, and it did not appear in the E. coli periplasm. We constructed a map of pVA318 for restriction endonucleases HindIII, HpaI, PstI, and HaeIII. A previously reported BamHI site in pVA318 did not appear in the pVA318 portion of any of our chimeric clones.

Cloning, Molecular↗

PubMed1990

Characterization of the F-plasmid conjugative transfer gene traU.

We characterized the traU gene of the Escherichia coli K-12 conjugative plasmid F. Plasmids carrying segments of the F transfer operon were tested for their capacity to complement F lac traU526. The protein products of TraU+ clones were identified, and the nucleotide sequence of traU was determined. traU mapped between traW and trbC. It encodes a 330-amino-acid, Mr36,786 polypeptide that is processed. Ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing of the protein to occur. Because F lac traU526 strains appear to be resistant to F-pilus-specific phages, traU has been considered an F-pilus assembly gene. However, electron microscopic analysis indicated that the traU526 amber mutation caused only a 50% reduction in F-piliation. Since F lac traU526 strains also retain considerable transfer proficiency, new traU mutations were constructed by replacing a segment of traU with a kanamycin resistance gene. Introduction of these mutations into a transfer-proficient plasmid caused a drastic reduction in transfer proficiency, but pilus filaments remained visible at approximately 20% of the wild-type frequency. Like traU526 strains, such mutants were unable to plaque F-pilus-specific phages but exhibited a slight sensitivity on spot tests. Complementation with a TraU+ plasmid restored the wild-type transfer and phage sensitivity phenotypes. Thus, an intact traU product appears to be more essential to conjugal DNA transfer than to assembly of pilus filaments.

Amino Acid Sequence↗

PubMed1996

Plasmid profiling for strain differentiation and characterization of Clostridium perfringens isolates.

Plasmid profiling was used for the characterization of 13 Clostridium perfringens collection strains as well as 85 clinical and food isolates. Methodological details and limitations of the plasmid isolation procedure are outlined and discussed. For 19 clinical isolates obtained during seven group outbreaks, a close connection between at least some strains from each individual outbreak was established for six of them; only results from one outbreak were completely inconclusive, due to missing plasmids in one of the two isolates. The presence of multiple plasmid types within seven out of 12 given food samples, from which at least two C. perfringens isolates were obtained, indicates the importance of multiple isolates for meaningful typing results in epidemiological investigations. By including results from a previous report from this laboratory, baseline data on plasmid profiles for a total of 133 isolates are provided. The results of this study revealed that 36% of the food isolates unrelated to disease outbreaks carried no plasmids, as compared with 19-25% among disease-related isolates. A high prevalence (24.8%) of a 8.9 +/- 0.5 MDa plasmid was found among the 133 isolates, which contributed to one of four occurrences of identical plasmid profiles among strains that were initially considered unrelated. Two of these identical plasmid profiles were found among strains from the same culture collection, indicating the possibility of a common ancestor strain or cross-contamination.

Animals↗

PubMed1984

Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.

Bacterial Outer Membrane Proteins↗

PubMed1979

Construction and characterization of E. coli promoter-probe plasmid vectors. I. Cloning of promoter-containing DNA fragments.

Derivatives of the Escherichia coli drug-resistance plasmid pBR316 have been constructed which act as molecular probes for promoter-containing DNA restriction fragments from various prokaryotic genomes. The plasmids, designated pBRH1 and pBRH3B, contain a unique EcoRI restriction site located within the promoter for the tetracycline resistance (Tcr) gene. This site was created by the insertion of a chemically synthesized octanucleotide, containing the EcoRI cleavage sequence, into the HindIII site of pBR316. Base-pair alterations within the Tc promoter produced by this insertion resulted in a substantial reduction (pBRH3B) or elimination (pBRH1) in ability of these plasmids to confer Tc resistance to the host strain. Cloning of EcoRI-cleaved foreign DNA fragments into the EcoRI site of these plasmids allows for the isolation of recombinant transformants with Tcr levels greater than that of the plasmid vector. Further characterization of these recombinant plasmids demonstrates that the Tcr phenotype is dependent upon the orientation of the inserted fragment, but not on the molecular weight. We have concluded that these fragments carry promoters which, in the proper orientation, allow for the transcription of the Tcr gene. The utility of these "promoter-probe" plasmids lies in the ability to select for promoter-containing DNA fragments by insertional activation of the Tcr gene.

Bacillus subtilis↗