Microbiological implications of electric field effects VII. Stimulation of plasmid transformation ofBacillus cereus protoplasts by electric field pulses

Shivarova, N.; Förster, W.; Jacob, H.‐E.; Grigorova, R.

doi:10.1002/jobm.3630230915

Cited by 69 publications

(20 citation statements)

References 10 publications

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“…Since this technique has been successfully applied to mammalian cells, plant protoplasts, yeast, and, now, bacterial cells, it most likely depends on conserved physical properties of biomembranes. In addition, the transformation frequency of highly competent Bacillus cereus protoplasts with a Bacillus thuringiensis plasmid has been shown to be moderately increased by application of an electric pulse (22). Electroporation may therefore be a general method that will be useful for introducing DNA into many bacterial species in addition to C. jejuni and C. coli (see below).…”

Section: Discussionmentioning

confidence: 99%

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Miller

Dower

Tompkins

1988

Proc. Natl. Acad. Sci. U.S.A.

191

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Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacterjejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 X 106 transformants per #g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.Electroporation involves the application of high-intensity electric fields of short duration to reversibly permeabilize biomembranes. This technique is commonly used to transfer DNA into mammalian cells (1)(2)(3)(4)(5)25), and applications for plant protoplasts and yeast have been reported (6-9). Electrical impulses have also been shown to greatly increase the frequency of eukaryotic cell fusion events (10). Since electroporation may depend on fundamental properties of biological membranes, it appeared feasible to use this technique to promote genetic transformation of intact Campylobacter jejuni cells.C. jejuni, a member of the genus Campylobacter that contains Gram-negative, microaerophilic spiral-shaped bacilli, is a major cause of human diarrheal disease throughout the world (11, 12). Considerable effort is therefore being directed toward understanding the molecular basis of pathogenesis of this species. Genetic analysis of C. jejuni and its putative virulence factors has been difficult due to the absence of characterized systems for in vivo genetic manipulation (13 Plasmid pILL512 (14) is a pBR322 replicon containing the cryptic C. coli plasmid pIP1445 (17), the aphA-3 kanamycinresistance determinant (16), and the RK2 oriT sequence (18). pILL512 was introduced into C. jejuni C31 by mobilization in trans from an HB101 strain containing pILL512 and the Tra+ RK2 derivative pRK212-1 (19). Matings were performed as described (14). Plasmid KH2PO4, pH 7.4) and pelleted at 6000 x g for 8 min at 20°C. Cells were gently resuspended in EPB to a density of -5 x 109 cfu/ml. pILL512 DNA isolated from C31 and purified by cesium chloride/ethidium bromide equilibrium gradient centrifugation was then ad...

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Section: Discussionmentioning

confidence: 99%

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Miller

Dower

Tompkins

1988

Proc. Natl. Acad. Sci. U.S.A.

191

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“…Electroporation has been used to transform bacterial (15) and mammalian (6) cells with high efficiency (>10-4) and should be equally effective with plant cells. Recently, Fromm et al (16) reported the introduction of foreign genes into maize protoplasts by electroporation, and Shillito et al (17) reported high-efficiency transformation of plants when electroporation was used in combination with PEG, Mg2+, and a heat shock treatment.…”

mentioning

confidence: 99%

Stable transformation of tobacco by electroporation: evidence for plasmid concatenation.

Riggs¹,

Bates²

1986

Proc. Natl. Acad. Sci. U.S.A.

122

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Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence ofthe linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 x 10-(based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-pis-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots ofDNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head-to-head and head-to-tail concatemers in most ofthe plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that-the kanamycinresistance marker is inherited as a single dominant gene.

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“…2 ) Recently gene transfer was obtained with walled systems, such as bacteria 3) and yeasts,4) and electroporation is, in fact, now routinely used in many bacterial genetics laboratories. Especially, since an early study by Shivarova et al 5 ) electrotransformation of the genus Bacillus with plasmid DNAs, such as pGK12 with 4.4 kbp, 6) pC 194 with 2.9 kbp, 7 ,8) pBAM 101 with 6.8 kbp,9) and pUBIIO with 4.5 kbplO) was done. Bacillus strain produces a large variety of extracellular enzymes, some of which are of industrial importance, and offers many potential advantages in the production of cloned gene products.…”

mentioning

confidence: 99%

Effects of Plasmid DNA Sizes and Several Other Factors on Transformation ofBacillus subtilisISW1214 with Plasmid DNA by Electroporation

Ohse

Takahashi

Kadowaki

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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Microbiological implications of electric field effects VII. Stimulation of plasmid transformation ofBacillus cereus protoplasts by electric field pulses

Cited by 69 publications

References 10 publications

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Stable transformation of tobacco by electroporation: evidence for plasmid concatenation.

Effects of Plasmid DNA Sizes and Several Other Factors on Transformation ofBacillus subtilisISW1214 with Plasmid DNA by Electroporation

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