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Results for “plasmid characterization”

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At least 19 recordsLinked to original sources
PubMed1978

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.

DNA, Bacterial↗
PubMed1979

Physical mapping of Staphylococcus aureus penicillinase plasmid pI524: characterization of an invertible region.

The staphylococcal penicillinase plasmid pI524 and a series of derivatives have been extensively mapped by restriction endonuclease digestion and by heteroduplex analysis. We report here the identification of a 2.2 kb region that undergoes a reversible, rec-independent inversion. This sequence is bounded by a pair of inverted repeats 650 base pairs in length, and has asymmetrically located recognition sites for at least three restriction endonucleases. A series of deleted derivatives and one naturally occurring, closely related plasmid, were studied. Two of these retain the inversion; the remainder are incapable of inverting and were all found to be locked in the same orientation of the inversion. The invertible sequence is adjacent to the region of the plasmid encoding beta-lactamase (bla); this entire region appears to be transposable and the inversion may be involved in the regulation of beta-lactamase expression or in translocation.

DNA Restriction Enzymes↗
PubMed1979

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.

Animals↗
PubMed1978

Characterization of plasmids that encode for the K88 colonization antigen.

K88 antigen, and important virulence factor in porcine enteropathogenic Escherichia coli (EEC), can be transferred along with the ability to ferment the trisaccharide raffinose (Raf). The plasmids from a number of EEC strains that encode these two properties were isolated and characterized. In most strains the K88 and Raf genes were found on a single nonconjugative plasmid approximately 50 x 10(6) daltons in size. This plasmid core was conserved with only slight variation among the strains tested. In some transconjugants, larger conjugative plasmids were observed that were apparently recombinants between the Raf/K88 plasmid and a transfer fa(tor. Occasionally plasmids carrying only the raffinose fermentation genes arose by deletion of a deoxyribonucleic acid segment of about 20 x 10(6) daltons that included the K88 antigen gene(s).

Animals↗
PubMed1978

[Characterization of plasmid DNA in a lecithinase-positive and in a lecithinase-negative strain of Clostridium perfringens (author's transl)].

A non pathogenic variant of Clostridium perfringens and the wild type strain were characterized. The strains agreed in most of the biochemical properties, in susceptibility against antibiotics and in bacteriocin production. Contrary to the wild type the variant did not produce lecithinase and gelatinase. In deoxyribonucleic acid (DNA) of both strains centrifuged in cesiumchlorid-ethidiumbromide equilibrium there was found a satellite peak containing three distinct, covalently closed circular (CCC) DNA elements. The sum of the average molecular weight or contour length of the two small circular molecules was equal to the average molecular weight or contour length of the third. The presence of the plasmids in the variant indicated that the synthesis of lecithinase might not be coded by a plasmid.

Bacteriocins↗
PubMed1979

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (E) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10(3) to 10(4) DNA molecules per transformant. Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.

Bacillus subtilis↗
PubMed1977

Characterization of plasmids determining hemolysin and bacteriocin production in Streptococcus faecalis 5952.

Two plasmids designated pOB1 and pOB2 were isolated from Streptococcus faecalis strain 5952 and found to have molecular weights of approximately 46 X 10(6) and 28 X 10(6), respectively. pOB1 was found to determine hemolytic activity and was transmissible, whereas pOB2 appeared to determine a bacteriocin that is specifically inhibitory to S. faecalis strains harboring the 26-megadalton plasmid pAM539.

Bacteriocins↗
PubMed1978

Characterization of plasmids in bacteriocin producing strains of Streptococcus faecium.

Four bacteriocin (enterocin) producing strains of Streptococcus faecium were examined for extrachromosomal DNA. Three strains proved to harbor minicircular plasmid DNA of uniform length as evidenced by CsCl ethidium bromide density centrifugation and electron microscopy. Molecular weight determination obtained through contour length measurements showed 2.4 X 10(6) dalton for S. faecium 25, 3.4 X 10(6) dalton for S. faecium 3 and 3.8 X 10(6) dalton for S. faecium 59. Elimination experiments for bacteriocin production with intercalating dyes proved sterile. The role of these plasmids remains for the time being unknown.

Anti-Bacterial Agents↗
PubMed1979

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes.

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.

Animals↗
PubMed1978

Isolation and preliminary characterization of R6K plasmid deletion mutants.

Several deletion mutants of R6K have been isolated by mutagen treatment of bacterial host carrying wild type of the plasmid and search for clones that lost ampicillin or streptomycin resistance. The molecular weight of the mutants as estimated by agarose gel electrophoresis was 15 times 10(6) minus 23 times 10(6) compared to 26 times 10(6) for the parental plasmid. The mutants were characterized in respect of the level of resistance to ampicillin and frequency of conjugational transfer. Some of the mutants were found to differ in Eco RI digestion pattern from the wild type.

Ampicillin↗
PubMed2026

Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of Clostridioides difficile and Clostridium perfringens.

UNLABELLED: Clostridioides difficile and Clostridium perfringens are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two C. difficile and two C. perfringens strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for C. difficile, achieving >100× coverage and enabling reliable circularization of chromosomes and plasmids, including a C. difficile metronidazole resistance plasmid and C. perfringens plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for C. difficile, highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of C. difficile and C. perfringens, supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens. IMPORTANCE: High-quality genomic data are essential for accurate characterization of Clostridioides difficile and Clostridium perfringens, two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of C. difficile and C. perfringens for genomic surveillance or outbreak investigations.

Clostridioides difficile↗
PubMed2026

Phylogenetic distribution and longitudinal persistence of plasmids in Mycobacterium abscessus.

Mycobacterium abscessus, a non-tuberculous mycobacterium, is a cause of severe respiratory infections, notably in individuals with underlying lung conditions. Its high levels of intrinsic and acquired antimicrobial resistance make it particularly difficult to treat and horizontally acquired genetic elements may facilitate the spread of resistance. A small number of plasmids have been identified in this species, but their distribution, transmission dynamics across subspecies and clonal lineages remain poorly characterized. We analysed short-read genomic data from 3,060 M. abscessus isolates, including longitudinal samples, to characterize plasmid diversity and dynamics. Using a graph-based pan-genome approach, we identified 28 plasmids, including 15 previously unreported plasmids, mapped their distribution onto the species phylogeny and assessed their functional potential. Overall, 23.1% of isolates carried at least one plasmid, with higher prevalence in dominant circulating clones (DCCs) compared with non-DCCs. Plasmid carriage varied across subspecies and clonal backgrounds, and plasmids encoded numerous genes which may be linked to bacterial adaptation. Several plasmids persisted across multiple time points within individual patients, suggesting they can be highly stable over the course of a chronic infection.

Plasmids↗
PubMed1978

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus wigh high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site and two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).

Bacillus subtilis↗
PubMed1977

Physical characterization of a plasmid cointegrate containing an F'his gnd element and the Salmonella typhimurium LT2 cryptic plasmid.

A recombinant plasmid (pAS19) isolated from a derivative of Salmonella typhimurium LT2, containing the strain LT2 cryptic plasmid and an F'his gnd element, has been physically characterized. The pAS19 plasmid contour length equals the sum of the contour lengths of the cryptic plasmid and F'his gnd element. Deoxyribonucleic acid (DNA)-DNA hybridization experiments demonstrated that whereas the pAS19 plasmid exhibits extensive DNA homology with both the cryptic plasmid and the F'his gnd element, there is little DNA homology between these latter two plasmids. The DNA fragmentation pattern of the pAS19 plasmid produced by the restriction endonuclease R-EcoRI is consistent with that expected for a composite plasmid cointegrate containing most, if not all, of the DNA sequences present in its two component plasmids.

Animals↗