Plasmid Loss in Plasmid-carrying Strains of Escherichia coli Treated with Phenoxazines and an Approach to Study Their DNA Binding Properties

The effect of subinhibitory concentrations of 2-trifluoromethyl-N10-substituted phenoxazines on plasmid-coded antibiotic resistance in Escherichia coli was investigated. Phenoxazine treatment resulted in the loss of resistance markers to an extent of 8-63% in all the strains tested, and the disappearance of plasmid DNA in phenoxazine sensitive colonies was evidenced by agarose gel electrophoresis. The resistant strains were sensitized in the presence of phenoxazines with a concomitant reduction in the MIC (minimum inhibitory concentration) values. The UV, fluorescence spectral, and ethidium bromide displacement agarose gel assay methods revealed that phenoxazines are intercalated with plasmid DNA. Progressive addition of DNA led to a significant reduction in the peak intensity of the absorption maximum of phenoxazine derivative. Further, destabilization of ethidium bromide-DNA complex as seen from fluorescence microscopy in the presence of phenoxazines was observed. The potency of phenoxazines to sensitize the resistant organisms follows the order butyl > propyl > acetyl derivatives.     

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N¹⁰‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid _PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Interaction of 2-chloro-N10-substituted phenoxazine with DNA and effect on DNA melting

Five N10-substituted phenoxazines having different R groups and -Cl substitution at C-2 were found to bind to calf -thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA-ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.     

Decreased resistance to antibiotics and plasmid loss in plasmid-carrying strains of Staphylococcus aureus treated with ascorbic acid

The effect of ascorbic acid on plasmid-coded antibiotic resistance in Staphylococcus aureus was investigated. Several strains of S. aureus were cultured in the presence of 1 mM ascorbate for 6 h. This treatment induced an increased loss of resistance markers in 4 of 6 strains tested, and agarose gel electrophoresis showed this disappearance of plasmid DNA in ascorbate-induced susceptible colonies. The presence of ascorbate induced a 50-75% decrease in minimal inhibitory concentrations of different antibiotics for resistant strains. When ascorbate is added, formerly subinhibitory concentrations of penicillin or tetracycline have an increased inhibitory effect on resistant strains and even induced the death of 25-93% of the initial population. These results suggest that ascorbate can induce the loss of several plasmids of S. aureus, and that the levels of antibiotic resistance are also affected by the presence of this compound.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Determination of plasmid copy number by fluorescence densitometry

A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.     

Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica)

Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica , were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents. Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin. Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%). While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63·4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11. Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4·3×10⁻³ transconjugants per donor cell.     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

Corrected Version Distribution of Heterogeneous and Homologous Plasmids in Bacillus spp

A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis, and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 mug/ml), sodium dodecyl sulfate (0.2 mug/ml), or novobiocin (1 mug/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens, S294, was found to harbor a plasmid, pFTB14, which differed from the plasmid species of classes 1 to 6 in B. subtilis and B. amyloliquefaciens, as determined by restriction analysis and DNA contour length determination. However, in DNA-DNA hybridization on a filter after Southern blotting from an agarose gel, the pFTB14 DNA hybridized with plasmids of classes 1 to 5. Three strains of B. thuringiensis each carried at least 4 to 11 plasmid species, whereas no plasmids were detected in four strains of B. cereus, which, in relation to B. thuringiensis, is closely related taxonomically and has highly homologous DNA sequences. The plasmid DNAs prepared from species other than B. subtilis and B. amyloliquefaciens did not hybridize with that of pFTB14.     

Comparison of plasmids in strains of Zymomonas mobilis

Four strains of Zymomonas mobilis were examined for their resistance to antimicrobial agents and found to have similar resistance profiles. Plasmid DNA was extracted and purified by CsCl dye-buoyant density centrifugation; molecular weights were determined by agarose gel electrophoresis and electron microscopy. All four strains harbored a large plasmid (46 X 10(6) Da) and a smaller plasmid (16-21 X 10(6) Da) whose molecular weight was strain dependent. Two strains, Ag11 and ATCC 10988, had smaller plasmids of unique molecular weight. Homology existed between the plasmids in the four strains as shown by cross-reaction in DNA-DNA blot hybridizations. Only one plasmid appeared unique to the host from which it was isolated.     

Expression of a plasmid borne ethidium resistance determinant from Staphylococcus in Escherichia coli: Evidence for an efflux system

Abstract A 1.1-kilobase (kb) DNA fragment derived from a staphylococcal plasmid specifying resistance to ethidium has been cloned into plasmid vector pUC9 in Escherichia coli . This fragment conferred resistant to ethidium and also to cetyltrimethyl ammonium ion in this host. Studies with labelled ethidium indicated that the basis of resistance was extrusion from the cell by an efflux system.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome.

Transposon Tn5 was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by conjugal transfer of the plasmid to Escherichia coli. Tn5 insertions were obtained by culturing L. pneumophila at the nonpermissive temperature (43 degrees C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked Tn5-containing strains, and Southern hybridizations indicated that Tn5, but not pRK340, inserted into multiple sites in the Legionella chromosome. In addition, the streptomycin resistance determinant on Tn5 was expressed in L. pneumophila.     

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 μmol/l ethidium bromide (EB) and then exposed to 110 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.     

Application of agarose gel electrophoresis to the characterization of plasmid DNA in drug-resistant enterobacteria.

A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.