Biological effects of stevioside on the survival of Escherichia colistrains and plasmid DNA

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250–300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.     

Phenotypic variations in strain AB1157 cultivars of Escherichia coli from different sources.

The purpose of this note is to alert users of Escherichia coli AB1157 and its derivatives to a potentially significant difference in cultivars from various sources. The difference we find is in the ability to host an infection by coliphage 186 after UV irradiation of the host cell.     

Mutagenic effects of gamma-rays on plasmid DNA in Escherichia coli

A purified and dried DNA of plasmid pKO482 (galK+) is 10 times more resistant to the inactivating action of 60Co-gamma-rays than that of lambda phage. gamma-Irradiation of the plasmid DNA induces forward mutations of galK, the frequency of which increases linearly with the dose. The efficiency of the mutagenic action of gamma-rays on the plasmid galK locus is 10(-12) per 1 rad and per 1 base pair. The mutagenic effect of gamma-radiation but slightly depends upon bacterial recA+ gene and upon the SOS-repair system induced by UV-irradiation of the recipient cells. It is assumed that the premutational lesions induced in the purified DNA by the direct effect of gamma-radiation are fixed into mutations by misreplication.     

Difference in frequency spectrum of extremely-low-frequency effects on the genome conformational state of AB1157 and EMG2 E. coli cells

The effect of weak extremely-low-frequency (ELF) magnetic fields (sinusoidal, 30 μT amplitude) on the genome conformational state (GCS) of E. coli mutant and wild type cells was studied by using the method of anomalous viscosity time dependency (AVTD) in the 6–37 Hz frequency range. We confirmed the existence of three resonance frequencies of 8.9, 15.5, and 29.4 Hz when mutant cells of K12 AB1157 strain were exposed. In the same frequency range, the wild type K12 EMG2 cells displayed only two effective windows, with resonance frequencies of 8.3 and 27 Hz. The resonance frequencies differed significantly (P < .001–.000001) in the strains studied, whereas other resonance parameters did not. It was concluded that mutations in the AB1157 strain resulted in a significant rearrangement in the ELF action spectrum, including the appearance of a new resonance. © 1996 Wiley-Liss, Inc.     

Lack of additivity of the effects of nonmutagenic stresses on the resistance ofEscherichia coli AB1157 to UV radiation

Previous work from this and other laboratories has shown that many unrelated, nonmutagenic physical, chemical, or nutritional stresses increase the resistance ofEscherichia coli AB1157 to short-wavelength UV while reducing their UV-induced mutation frequency. The mechanism appears similar in all these cases, suggesting that induction of the same error-free DNA repair pathway is involved. We now show that a combination of two such stresses is no more effective than one alone, in agreement with this hypothesis.     

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.     

Degradation of Escherichia coli chromosomal and plasmid DNA in serum

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.     

Topological alteration of plasmid DNA during cell growth of Escherichia coli.

Superhelical alteration of plasmid DNA in Escherichia coli cells was observed during cell growth. Plasmid DNAs were being supercoiled while the cells were actively dividing, and they were partially relaxed after the cultures reached stationary. Unknown structural materials of plasmid DNA which migrated faster than usual superhelices in agarose gel electrophoresis were found among the strains regardless of gyrase genotype.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli

In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.     

Membrane-bound fractions of R6K plasmid DNA in Escherichia coli.

The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.