Fusion of Erwinia chrysanthemi spheroplasts in an electric fields

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.     

Fusion of plant protoplasts by electric fields

The electrical fusion technique of Zimmermann and Scheurich (1981 Planta 151: 26-32) has been used to fuse mesophyll protoplasts of Avena, Zea, Vigna, Petunia, and Amaranthus. Electrical fusion proves to be a simple, effective, and general fusion technique that can be controlled to form either dikaryons or large multinucleate fusion bodies. In addition, we show that Vigna mesophyll protoplasts that are subjected to the electrical fields used in this technique are viable in culture. The construction of the fusion chambers, necessary electrical equipment, and the fusion protocol are described in sufficient detail for reproduction of the technique.     

Fusion of yeast spheroplasts.

Spheroplasts of two different auxotrophic strains of Saccharomyces cerevisiae, both of mating type a, were fused with the aid of polyethylene glycol and calcium ions. After reversion to vegetative cells in solid media, the resulting zygotes were shown to be diploid cells of mating type aa.     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

The influence of electric fields on bacterial growth

Growth of S.MARCESCENS (on TS agar) and of E.COLI (on EMB agar) was examined at various constant field strengths (growth being expressed by the diameter of colonies after incubation at 37°C for 46 hr). In the case of S. MARCESCENS the field strengths were calculated at 0, + or ÷ 940, and + or ÷ 3,125 v/cm;in the case of E.COLI at 0 and + or ÷ 3,125 v/cm. In experiments using S. MARCESCENS the mean diameters of colonies were found to be significantly reduced only at afield strength of + 940 v/cm as compared with diameters seen under field-free conditions. In experiments using E.COLI the mean diameters of the colonies were found to be significantly reduced at field strengths of + and ÷ 3,125 v/cm as compared with the diameters seen under field-free conditions.

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Zusammenfassung Das Wachstum von S.MARCESCENS (auf TG agar) und E.COLI (auf EBM agar) wurde bei verschiedenen konstanten Feldstärken untersucht. Für S. MARCESCENS wurden die Feldstärken berechnet bei 0, + oder 940 und + oder ÷ 3.125 v/cm;für E. COLI bei 0 und + oder ÷ 3.125 v/cm. Der mittlere Durchmesser der S.MARCESCENS Kolonien war nur bei der Feldstärke + 940 v/cm signifikant vermindert;der mittlere Durchmesser der E.COLI Kolonien war bei den Feldstärken von + und ÷ 3.125 v/cm signifikant vermindert, beide bezogen auf das Wachstum unter feldfreien Bedingungen.

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Resume On a observé la vitesse de croissance de S.MARCESCENS (sur Agar TS) et de E.COLI (sur Agar EBM) et cela dans divers champs électriques constants(la croissance était déterminée par le diamètre des colonies après une incubation de 46 heures à 37 °C). Pour S.MARCESCENS on a utilisé des champs de 0, + ou + 940 et + ou ÷ 3.125 v/cm. Pour E.COLI, les champs furent de 0 et de + ou ÷ 3.125 v/cm.Le diamètre moyen des colonies de S.MARCESCENS n'a été réduit de façon significative que pour un champ de + 940 v/cm. Le diamètre moyen des colonies de E.COLI a diminué de façon significative pour des champs de + ou ÷ 3.125 v/cm. Dans les deux cas, la diminution se rapporte aux conditions de croissances déterminées en l'absence de champ électrique.

     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles. Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.     

Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.     

Killing of microorganisms by pulsed electric fields

 Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (>99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (E _c) and a specific critical treatment time (t _c) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The “limits”E _c and t _c depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells. Receivced: 1 June 1995 / Received last revision: 13 September 1995 / Accepted: 20 September 1995     

Lysis of bacterial protoplasts and spheroplasts and suppression of their dehydrogenase activity by neotehomycin]

Neotelomycin induced lysis of the protoplasts of Bac. megaterium and inhibited their succinate dehydrogenase activity. Direct correlation between the lytic activity of the antibiotic and its effect on succinate dehydrogenase was found. Neotelomycin had no effect on the dehydrogenase activity of the protoplast lysates. Possibly, suppression of the protoplast succinate dehydrogenase of Bac. megaterium under the effect of neotelomycin was due to significant structural changes caused by the antibiotic in the protoplast membranes and leading to their lysis and not to the direct effect on the enzyme. Neotelomycin had practically no effect on the spheroplast dehydrogenase activity of E. coli resistant to the antibiotic and did not induce their lysis. Resistance of E. coli to neotelomycin must be associated not with the presence of the antibiotic non-permeable cell wall but the peculiar properties of the membrane cytoplasm.     

Anisotropic electric conductivity of bacterial suspensions induced by external electric field

The anisotropy of electrical conductivity of suspensions of such bacteria, as E. coli, Serratia marcescens, Pseudomonas fluorescens induced by a sinusoidal external electric field and relaxation of the anisotropy after switching off the field were investigated. On the basis of the experimental relationships the anisotropy of electrical polarizability and coefficient of rotational diffusion of the cells were evaluated. The anisotropy of electrical polarizability and coefficient of the rotational diffusion obtained are in a good agreement with the available data of other methods.     

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

Miniature-probe measurements of electric fields induced by 60 Hz magnetic fields in rats

Extremely-low-frequency (ELF) magnetic fields interact with an animal by inducing internal electric fields, which represent the internal dose from an external exposure. In this study, an electric field probe of approximately 2 mm resolution was used to measure fields induced in rat carcasses by a 60 Hz magnetic field at 1 mT. With the rat lying on its side, the probe was inserted through a small hole in the body wall, and scanned at 5 mm increments from the side with frontal and axial exposure (field horizontal) and from the front with lateral exposure (field vertical). The induced electric field declined from a maximum at the entrance to the abdomen and crossed zero to negative (180° phase shift) values within the body as expected. In general, the magnitudes of the measurements inside the abdomen were less than expected from whole-body calculations that used homogeneous-ellipsoidal models of a rat in the three orientations. The low measurements did not appear to be explained by perpendicular field components, by conductivity differences between the tissue and the probe path, or by air in the lungs. The low measurements probably result from inhomogeneities in actual rats that include conductivity differences between tissues and biological membranes. For example, an alternative model considered the abdominal cavity to be electrically isolated from the body by the diaphragm and the peritoneum and calculations from this model were in better agreement with the measurements inside the abdomen (than were the whole-body calculations). Therefore, inhomogeneities in conductivity and biomembranes such as the peritoneum should be considered in order to fully understand ELF-induced field dosimetry. © 1996 Wiley-Liss, Inc.     

Morphological transitions of vesicles induced by alternating electric fields

When subjected to alternating electric fields in the frequency range 10²-10⁸ Hz, giant lipid vesicles attain oblate, prolate, and spherical shapes and undergo morphological transitions between these shapes as one varies the field frequency and/or the conductivities λ_in and λ_ex of the aqueous solution inside and outside the vesicles. Four different transitions are observed with characteristic frequencies that depend primarily on the conductivity ratio λ_in/λ_ex. The theoretical models that have been described in the literature are not able to describe all of these morphological transitions.     

Confinement and manipulation of actin filaments by electric fields

When an AC electric field was applied across a small gap between two metal electrodes elevated above a surface, rhodamine-phalloidin-labeled actin filaments were attracted to the gap and became suspended between the two electrodes. The variance of each filament's horizontal, lateral displacement was measured as a function of electric field intensity and position along the filament. markedly decreased as the electric field intensity increased. Hypothesizing that the electric field induces tension in the filament, we estimated the tension using a linear, Brownian dynamic model. Our experimental method provides a novel means for trapping and manipulating biological filaments and for probing the surface conductance and mechanical properties of single polymers.