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PubMed1975

Construction and characterization of a chimeric plasmid composed of DNA Pfrom Escherichia coli and Drosophila melanogaster.

A chimeric plasmid has been constructed in vitro from colicin E1 factor (Col E1), nontransmissible R-factor RSF-1010, and Drosophila melanogaster DNAs by the sequential action of Escherichia coli endonuclease RI(Eco RI) and T4 phage DNA ligase. The chimeric plasmid was assembled in two stages--first, a composite plasmid consisting of Col E1 and RSF 1010 was constructed, followed by partial digestion of the composite with Eco RI (in order to open one of the susceptible cleavage sites) and ligation with an Eco RI-digested D. melanogaster DNA preparation. The chimeric plasmid was selected and amplified in vivo by sequential transformation of E. COLI C with the ligated mixture, selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-CsCl solution. Treatment of the chimeric plasmid with Eco RI yields three fragments with mobilities corresponding to the linear forms of the constituents--COL E1, mol wt 4.2 times 106, RSF 1010, mol wt 5.5 times 106 and D. melanogaster DNA, mol wt 4.0 times 106. The buoyant densities of the three constituents are respectively 1.706, 1.719, and 1.697 g/cm3, while the buoyant density of the composite factor is 1.712 and that of the chimeric plasmid is 1.705. Serratia marscesens endonuclease R (Sma) which introduces a single cut in Col E1, but not in RSF 1010, converts the chimeric plasmid to a single linear molecule (mol wt 13.7 times 106) and sequential digestion with both Sma and Hin III yields two distinct fragments, mol wt 3.7 and 10.0 times 10.6, respectively; this implies that the two sites are unique and occur at distinctly different positions. Sequential digestion with both Hin III and Eco RI reveals that the Hin III cut is in the D. melanogaster segment; neither Col E1 nor RSF 1010 contain sites susceptible to digestion with Hin III. In the presence of chloramphenicol, the chimeric plasmid continues toreplicate for 9 hr while bacterial chromosomal DNA replicates at a much slower rate. As in the case of the composite plasmid, continued synthesis is the presence of chloramphenicol suggests that the replicator of Col E1 is functional in the chimeric plasmid as well. Examination of the chimeric plasmid by partial denaturation mapping permits identification of its constituents, each of which presents a characteristic profile. The D. melanogaster segment reveals a wealth of detail at the molecular level pertaining to the distribution of AT-rich regions.

Animals↗

PubMed1999

Intracellular fate and nuclear targeting of plasmid DNA.

One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5-115 p-azidotetrafluorobenzyllissamine-NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide-NLS and plasmid lissamine-NLS conjugates interacted specifically with the NLS-receptor importin alpha. Plasmid-lissamine-NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid-lissamine-NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus.

3T3 Cells↗

PubMed2002

Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes.

PURPOSE: A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. METHODS: AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. RESULTS: Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. CONCLUSIONS: These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.

Animals↗

PubMed2003

Optimizing alkaline lysis for DNA plasmid recovery.

Optimization of the alkaline lysis (P2) and neutralization (N3) steps in the recovery of DNA plasmids was pursued. Experiments were conducted at the test-tube and 5-litre scales with 3 kb (pUC18) and 20 kb (pQR150) plasmids. The scale and degree of mixing/shear did not affect the optimum yield of supercoiled plasmid during the P2 step, but did effect the time required for the optimum to be achieved. This optimum time for P2 at the large scale was longer (8-9 min), especially when a low-shear impeller was used. Also, when the yield of supercoiled plasmid reached a maximum during the P2 step, the purity (percentage of plasmids in the supercoiled form) simultaneously reached a minimum. As the duration of the N3 step increased from 1 to 6 min, the yield of the supercoiled plasmids remained fairly constant, provided that a lowshear impeller was used. The neutralized (post-N3) plasmid solution was shear-sensitive; however, mixing with a Rushton turbine in a tank (maximum energy dissipation rate in the mixing tank, epsilon (max), 12 m(2)/s(3); mixing-tank power consumption/volume of mixing tank, 2.0 W/m(3)) for 5-10 min resulted in a slight decrease in supercoiled plasmid and a notable increase in genomic DNA concentrations. The loss of the larger 20 kb plasmid (20%) was more than for the 3 kb plasmid. Finally, preparing the cells for alkaline lysis with lysozyme or low-pressure homogenization did not increase the plasmid yield. Furthermore, the homogenizer broke up the genomic DNA into fragments that followed through the entire Qiagen prep with the plasmids as impurities.

Alkalies↗

PubMed2002

Delivery of plasmid DNA to articular chondrocytes via novel collagen-glycosaminoglycan matrices.

Our primary objective was to fabricate a porous gene-supplemented collagen-glycosaminoglycan (GSCG) matrix for sustained delivery (over a period of several weeks) of plasmid DNA to articular chondrocytes when implanted into cartilage lesions. The specific aims of this in vitro study were to determine the release kinetics profiles of plasmid DNA from the GSCG matrices, and to determine the ability of the released plasmid DNA to transfect adult canine articular chondrocytes. In particular, we evaluated the effects of two variables, cross-linking treatment and the pH at which the DNA was incorporated into the matrices, on the amount of the plasmid DNA that remained bound to the GSCG matrices after passive (nonenzymatic) leaching and on the expression of a reporter gene in articular chondrocytes grown in the GSCG matrices. Collagen-glycosaminoglycan matrices were synthesized without cross-linking, and by three cross-linking treatments: dehydrothermal (DHT) treatment, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) treatment, and exposure to ultraviolet (UV) radiation. The plasmid DNA was incorporated into the collagen-glycosaminoglycan matrices in solutions at pH 2.5 or 7.5. Transmission electron microscopy studies revealed plasmid DNA bound to the walls of the porous GSCG matrices. In general, the GSCG matrices fabricated at pH 2.5 retained a larger fraction of the initial DNA load after 28 days of incubation in Tris-EDTA buffer. The passive, solvent-mediated release of the plasmid DNA from the GSCG matrices showed a biphasic pattern consisting of a faster, early release rate over the initial 8 hr of leaching followed by a slower, late release rate that was relatively constant over the subsequent 28 days of leaching. Electrophoretic analyses revealed that the plasmid DNA released from the GSCG matrices fabricated at pH 2.5 had been linearized and/or degraded; whereas the plasmid DNA leached from the GSCG matrices prepared with a DNA solution at pH 7.5 was primarily supercoiled and linear. Plasmid DNA released from all GSCG matrix formulations was able to generate luciferase reporter gene expression in monolayer-cultured chondrocytes transfected with the aid of a commercial lipid reagent, and in chondrocytes cultured in the GSCG matrices without the aid of a supplemental transfection reagent. Luciferase expression in chondrocyte-seeded GSCG constructs was evident throughout the culture period (28 days), with the EDC and UV cross-linked matrices prepared at pH 7.5 providing the highest transgene expression levels. We conclude that released plasmid DNA continually transfected canine articular chondrocytes seeded into GSCG matrices in vitro for a 4-week period as evidenced by luciferase reporter gene expression. Thus, GSCG matrices can be fabricated to provide sustained release of plasmid DNA carrying a potential therapeutic gene.

Animals↗

PubMed1992

The relapsing fever agent Borrelia hermsii has multiple copies of its chromosome and linear plasmids.

Borrelia hermsii, a spirochete which causes relapsing fever in humans and other mammals, eludes the immune response by antigenic variation of the "Vmp" proteins. This occurs by replacement of an expressed vmp gene with a copy of a silent vmp gene. Silent and expressed vmp genes are located on separate linear plasmids. To further characterize vmp recombination, copy numbers were determined for two linear plasmids and for the 1-megabase chromosome by comparing hybridization of probes to native DNA with hybridization to recombinant plasmids containing borrelial DNA. Plasmid copy numbers were also estimated by ethidium bromide fluorescence. Total cellular DNA content was determined by spectrophotometry. For borrelias grown in mice, copy numbers and 95% confidence intervals were 14 (12-17) for an expression plasmid, 8 (7-9) for a silent plasmid, and 16 (13-18) for the chromosome. Borrelias grown in broth medium had one-fourth to one-half this number of plasmids and chromosomes. Staining of cells with 4',6-diamidino-2-phenylindole revealed DNA to be distributed throughout most of the spirochete's length. These findings indicate that borrelias organize their total cellular DNA into several complete genomes and that cells undergoing serotype switches do one or more of the following: (1) coexpress Vmps from switched and unswitched expression plasmids for at least three to five generations, (2) suppress transcription from some expression plasmid copies, or (3) partition expression plasmids nonrandomly. The lower copy number of the silent plasmid indicates that nonreciprocal Vmp gene recombination may result from loss of recombinant silent plasmids by segregation.

Bacterial Outer Membrane Proteins↗

PubMed1993

Telomere-mediated plasmid segregation in Saccharomyces cerevisiae involves gene products required for transcriptional repression at silencers and telomeres.

Plasmids that contain Saccharomyces cerevisiae TG1-3 telomere repeat sequences (TRS plasmids) segregate efficiently during mitosis. Mutations in histone H4 reduce the efficiency of TRS-mediated plasmid segregation, suggesting that chromatin structure is involved in this process. Sir2, Sir3 and Sir4 are required for the transcriptional repression of genes located at the silent mating type loci (HML and HMR) and at telomeres (telomere position effect) and are also involved in the segregation of TRS plasmids, indicating that TRS-mediated plasmid segregation involves factors that act at chromosomal telomeres. TRS plasmid segregation differes from the segregation of plasmids carrying the HMR E silencing region: HMR E plasmid segregation function is completely dependent upon Sir2, Sir3 and Sir4, involves Sir1 and is not influenced by mutations in RAP 1 that eliminate TRS plasmid segregation. Mutations in SIR1, SIN1, TOP1, TEL1 and TEL2 do not influence TRS plasmid segregation. Unlike transcriptional repression at telomeres, TRS plasmids retain partial segregation function in sir2, sir3, sir4, nat1 and ard1 mutant strains. Thus it is likely that TRS plasmid segregation involves additional factors that are not involved in telomere position effect.

Chromatin↗

PubMed1993

Plasmid suppressors active in the sexual cycle of Neurospora intermedia.

We have discovered that, in certain crosses of natural isolates of Neurospora intermedia, linear and circular mitochondrial plasmids of the maternal parent are not transmitted to the progeny. This contrasts with the maternal transmission of organellar genetic elements generally observed in crosses between laboratory strains and between other natural isolates. Formally, failure of plasmid transmission is a type of plasmid suppression. The present cases represent the first report of plasmid suppressors in natural populations of fungi. Strains used as female parents can transmit or not transmit plasmids depending on the strain used as male parent. Males that act to suppress in one cross fail to suppress in others. Therefore, the suppression of plasmids depends on a strain-specific interaction and is not determined exclusively by the males. Since suppression is a specific interaction we inferred that it must be genetically based and tested this hypothesis by seeking segregation of suppressed and nonsuppressed phenotypes in octads. Segregation of the original full suppression of all plasmids was indeed observed in each of the three sets of testcrosses examined. The interaction type of suppression must be initiated in ascogenous tissue during the sexual cycle. It is a nonautonomous type of suppression, affecting all descendent cells. In any one case of suppression, either one, several, or all plasmids can be lost. Both linear and circular plasmids can be eliminated by the same suppressor genotype. In addition, several strains were found to contain suppressors that act after ascospore delineation. This autonomous type of suppression has been observed previously in laboratory strains, but not in natural isolates. All the cases of plasmid suppression identified in this study involved a range of apparently neutral circular and linear plasmids. Using one senescent Kalilo strain of N. intermedia, we did not detect any case of suppression of the senescence-determining linear plasmid kalDNA.

Crosses, Genetic↗

PubMed1991

Molecular evolution of tetracycline-resistance plasmids carrying TetM found in Neisseria gonorrhoeae from different countries.

High level tetracycline resistant strains of Neisseria gonorrhoeae (TRNG) have been shown to carry a 40.6 kb (25.2 MDa) conjugative plasmid with a Class M tetracycline resistance determinant. Restriction endonuclease analysis mapping showed that there were at least two different TRNG plasmid types which were found in geographically distinct locations. The physical maps of these two plasmids were compared to a gonococcal conjugative plasmid which did not encode tetracycline resistance. The plasmid type which is endemic in the Netherlands was found to be closely related to the gonococcal conjugative plasmid, which supports the established hypothesis that the 40.6 kb plasmid has evolved by transposition of the TetM determinant into the conjugative plasmid. The plasmid found in the United States has either evolved by substantial divergent evolution or it results from a different transposition event. In the UK there have been isolations of TRNGs carrying either of the two plasmid types reflecting a flow of people both across the Atlantic and in Europe. It is possible that further TetM-containing plasmids will be found in N. gonorrhoeae paralleling the family of TEM beta-lactamase encoding plasmids already described.

Bacterial Proteins↗

PubMed2006

Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia.

OBJECTIVES: To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. METHODS: Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. RESULTS: Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed bla(CMY-7) on an approximately 93 kb plasmid. In CG 1 isolates, bla(TEM), catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an approximately 170 kb plasmid. In CG 2 isolates, a second approximately 93 kb plasmid contained bla(TEM) and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the bla(CMY-7)-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the bla(TEM)/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. CONCLUSIONS: This is the first report on the detection of plasmid-mediated bla(CMY-7) in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.

Animals↗

PubMed2006

Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom.

OBJECTIVES: The UK, like other countries worldwide, has a growing problem with CTX-M beta-lactamase-producing Escherichia coli. Five major clonally related strains have been identified among CTX-M-15 producers. We characterize here the plasmids from clonal strains A and D. METHODS: Plasmids were extracted and transformed into E. coli DH5alpha; conjugative mating was attempted on agar. MICs were determined by agar dilution. beta-Lactamases were typed by isoelectric focusing; antibiotic resistance genes and integrons were identified by PCR and sequenced. Plasmid incompatibility groups were determined by replicon PCR. RESULTS: bla(CTX-M-15) was carried by a 150 kb plasmid in strain A and a 70 kb plasmid in strain D. Conjugative transfer of cefotaxime resistance was only achieved from strain D; plasmids from both strains were transferred by transformation. The plasmid from strain A additionally carried bla(TEM-1) (variably), bla(OXA-1), aac(6')-Ib-cr and tet(A), as well as a class 1 integron with the gene cassettes aadA5 and dfr(17); the plasmid from strain D carried bla(TEM-1) consistently, also bla(OXA-1), aac(6')-Ib-cr, aac3-IIa and tet(A). Both plasmids belonged to incompatibility group FII. CONCLUSIONS: bla(CTX-M-15) was plasmid-mediated in both strains A and D and was linked to other antibiotic resistance genes including aac(6')-Ib-cr, which encodes an acetyltransferase, not previously found in Europe, acting on both aminoglycosides and some fluoroquinolones. Although the plasmids from the two strains differed in size, both were related and conferred similar multi-drug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 beta-lactamases in E. coli from Canada and India.

Anti-Bacterial Agents↗

PubMed2005

The evolution of a conjugative plasmid and its ability to increase bacterial fitness.

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.

Biological Evolution↗

PubMed1980

Studies of temperature-sensitive transfer and maintenance of H incompatibility group plasmids.

The mechanism of temperature-sensitive transfer was studied for plasmids of the H incompatibility group. Transfer depended on the temperature of the mating mixture but the growth temperature of the donor was also important, and donor cells previously grown at 26 degrees C could not facilitate transfer at 37 degrees C. Comparison of transfer characteristics of a non-thermosensitive H plasmid R831b and thermosensitive H plasmids from Salmonella from Ontario during a 2 h mating period showed that the thermosensitive phenotype inhibited transfer by about 200-fold at 37 degrees C and by 10-fold at 42 degrees C. At temperatures between 15 and 30 degrees C, the thermotolerant H plasmid transferred at about the same frequency as the temperature-sensitive plasmid. Elimination of some H plasmids after growth of host cells was also observed and physical evidence of this was obtained. The characteristic of high-temperature elimination (Hte) was limited to plasmids from similar bacterial and geographphical sources. Plasmids from Salmonella spp. isolated in Ontario did not possess this phenotype, whereas plasmids from Serratia marcescens isolated in the United States did. Although the Tra(ts) and Hte phenotypes may both be characteristic of H plasmids, they were shown to be separate and distinct properties. The H plasmids used in this study were isolated and their molecular weights determined by agarose gel electrophoresis. All were large, with molecular weights often exceeding 140 X 10(6). In contrast, the thermostable H plasmid R831b had a molecular weight on only 49 X 10(6).

Conjugation, Genetic↗

PubMed1985

Growth inhibition of Escherichia coli by E colicin plasmids.

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.

Amino Acids↗

PubMed1986

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex.

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.

Adsorption↗

PubMed1986

No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains.

A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 microM-thallium salt and susceptibility to the nocardia-specific bacteriophage phi B1. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 X 10(-4) with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, phi B1s) and 2 X 10(-3) with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, phi B1r) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 microM-thallium acetate). The frequency of thallium-resistant transconjugants was 10(-1) to 10(-2) per donor; all thallium-resistant transconjugants contained the donor plasmid. We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.

Bacteriophages↗

PubMed1989

Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells.

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.

Adhesins, Escherichia coli↗

PubMed1990

Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus.

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.

Bacteriocins↗