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[Action of different types of gramicidin S derivatives on bacterial cells and protoplasts].

The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts. The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells. However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis. Hence, these compounds preserved a certain membranotropic level. Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts.

Bacillus megaterium↗

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts.

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.

Atmosphere Exposure Chambers↗

Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts.

The uptake and expression of cucumber mosaic viral (CMV) RNA by tobacco protoplasts was examined using both square wave and exponential wave electroporation pulses. These electropulses, when supplied at sufficient field strength for a critical duration, enabled RNA to be incorporated and expressed in more than 60% of the surviving protoplasts. The results of experiments using these two electroporation wave forms showed significant differences in RNA uptake and expression. The number of viable protoplasts and cells showing expression of RNA was higher over a much broader range of experimental conditions using the square rather than exponential wave generator, even when both machines were optimized for maximal performance. However, at a narrow range of low field strengths the exponential wave pulse generated a higher percentage of transformants than did the square wave pulse. It was shown that after an electroporation pulse from either wave form, there were viable cells which expressed foreign RNA at predictable levels.

Biotechnology↗

Binding of transforming DNA to protoplasts isolated from competent Bacillus subtilis.

Transforming 3H-labelled DNA binds specifically to protoplasts isolated from competent cultures of Bacillus subtilis. The bound DNA is fully accessible to added DNase I, indicating that protoplasts bind, but do not process donor DNA. A bi-phasic pattern of competition for binding with increasing amounts of unlabelled and labelled DNA was found. In these conditions, two levels of saturation appeared which correspond to two kinds of DNA receptor sites. Homologous DNA binds preferentially to the first kind of receptor (specific) and only at higher concentrations would bind to the second (unspecific) binding sites. Bound DNA forms a rather stable complex with some constituent(s) of the protoplasts.

Bacillus subtilis↗

Isolation, purification and regeneration of protoplasts from Sporotrichum thermophile conidiospores.

Protoplasts of uniform size were prepared from mononucleated conidiospores of Sporotrichum thermophile. Conidia were preincubated in glucose yeast extract medium at 45 C for 4 h. The conidia were collected resuspended in buffer containing 0.6 M KCl (as stabilizer), and incubated with Novozyme SP249 and Cellulase CP at 37 C for 6 h. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 50% sucrose. The purified protoplasts were regenerated in glucose yeast extract broth after 7 h of incubation at 45 C.

Cell Wall↗

[The protoplasts of Acremonium chrysogenum. Biochemical and morphological studies].

A procedure for preparing stable A. chrysogenum protoplasts capable of 60 per cent regeneration was developed. Two morphogenetic types of the regeneration were detected. The variants isolated after the protoplast regeneration were characterized by wide ranges of morphological variation. Capacity for the antibiotic production varied from 60 to 160 per cent of the activity of the starting strain. A procedure for isolating functionally active mitochondria from protoplasts of A. chrysogenum was also developed. Their main bioenergetic parameters were studied. In the respiratory chain of the A. chrysogenum mitochondria there were detected three conjugation sites of oxidative phosphorylation.

Acremonium↗

Protoplast formation and the visualization of nuclei in the eukaryotic microbe, Achlya.

Protoplasts have been released form 21 h germlings of the aquatic fungus, Achlya. The commercial enzyme preparation Driselase was utilized to digest the hyphal walls. Protoplast release began 20 min after incubation at 25 degrees C, and was completed by 2 hr after enzyme addition. The fluorescent dye mithramycin was utilized to observe nuclei both in tract hyphae and in isolated protoplasts.

Cell Nucleus↗

Effect of the composition of reversion medium on change of Staphylococcus aureus lysostaphin protoplasts to coccal forms and L-forms.

The experimental conditions under which protoplasts of Staphylococcus aureus strain MS353 (pCp) are converted to the coccal or L-form were investigated. Protoplasts prepared by treating coccal MS353 (pCp) strain with Lysostaphin formed various types of colonies (coccal form, L-form and mixed types) in about 50% yield when they were plated on reversion (R) medium consisting of 2% brain heart infusion, 0.5M sodium succinate, 0.01% bovine serum albumin, 20 mM MgCl2 and 0.6% agar. The L-form type colonies with a typical fried-egg appearance that developed on the R medium at an early stage gradually reverted to the coccal form through a mixed type stage in which a high density area first appeared in the periphery of the colony and then spread throughout the colony. The use of modified R medium without MgCl2 or R medium in which 0.5M sodium succinate as an osmotic stabilizer was replaced by 7.5% NaCl resulted in marked delay in the appearance of reverted cells. R medium without bovine serum albumin yielded atypical L-form type colonies, which contained masses of coccal cells with very irregular margins. On the other hand, R medium without MgCl2 but with penicillin G supported development of L-form type colonies at high rate (13-15%) from the inoculated protoplasts.

Culture Media↗

[Release of "Saccharomyces cerevisiae" protoplasts: scanning electron microscope study (author's transl)].

In a previous study we described the minimal methodology used to obtain protoplasts from ascomycetous yeasts. Using a reducing agent associated with 1,3-beta-glucanase at 26 degrees C, protoplasts were invariably obtained. In the present study we localized the disruption spots of the cell wall using the two same reagents. The observations were made with the scanning electron microscope. The disruption site was always in the subterminal region, and this very simple structure (proteins with disulphide bridges and 1,3-beta-glucans) was opposite the birth-scar. The dissociation of the two reagents showed that a small part of the yeast population was able to release protoplasts with only glucanase. We believe these very sensitive yeasts (2 to 10% population) to be very young cells. These disruption sites seemed very different from budding-sites. They might be identical with elongation-sites, or with the opening in the ascus-wall during germinating ascospore release.

Glucan Endo-1,3-beta-D-Glucosidase↗

Delivery of liposome content into plant protoplasts.

The interaction of various types of liposomes with protoplasts of Nicotiana tabacum and Capsicum annuum was studied by means of fluorescence microscopy and autoradiography. The liposomes with a positive charge displayed the strongest affinity to the protoplast membrane, whereas the maximum intracellular delivery of encapsulated protein and DNA molecules into protoplasts was exhibited by the negatively charged liposomes. The results showed that the negatively charged unilamellar liposomes prepared by the reverse-phase evaporation method represent a very promising universal vehicle system for introducing genetic information into plants.

Autoradiography↗

[New scanning electron microscopy contribution to the study of yeast protoplasts].

Study by scanning electron microscopy enables us to describe with precision the morphology of protoplasts. We can confirm that the wrinkles more or less perceptible on the protoplasts studied, do belong to the morphology of those just mentioned, that the presence or the absence of globulous component is connected with the physiological activity of the cell and that it is possible to distinguish the different protoplasts thanks to their morphological aspect.

Candida↗

[Protoplast isolation from a nisin-forming culture of Streptococcus lactis strain MGU].

A procedure for protoplasts of Str. lactis producing nisin was developed. The following lysing factors were used for obtaining the protoplasts: lycozyme, LE enzyme, lysosubtilin and an enzyme isolated from the culture fluid A. levoris. The use of the above factors in different combinations provided an increase in the number of the formed protoplasts from 1-2 in the field of the microscope vision to 15-20, which amounted to 25 per cent of the total number of the cells visible under microscope.

Lactococcus lactis↗

[Effect of polyene antibiotics on bacterial protoplasts].

The carbonyl-conjugated pentaenes flavofungin, nigrofungin and flavopentin exhibit considerable lytic activity toward Micrococcus lysodeikticus and Bacillus megaterium protoplasts. The antibiotics at concentrations of 5 to 14 microgram/ml cause lysis of 50% of the protoplasts within 15 min of their incubation. The antibiotics inhibit the activity of NADH oxidase and malate oxidase by 50% in the lysates of Micrococcus lysodeikticus and Bacillus megaterium protoplasts at concentrations of 30 to 50 microgram/ml; preincubation of the lysates with the antibiotics intensify the inhibiting action of the polyenes. Growth of the bacteria is inhibited when the minimal concentration of the polyenes is 75 to 100 microgram/ml. Interaction of the polyenes with bacterial membranes lacking sterols indicates that resistance of at least some bacteria to polyenes is caused by impermeability of the cell wall for these substances rather than by the absence of sterols in the membranes.

Anti-Bacterial Agents↗

[Comparative study of plasmid transformation in competent cells and protoplasts of Bacillus subtilis].

The effect of different factors which inhibit transformation with chromosomal DNA on plasmid transformation of competent cells and protoplasts was examined. The treatment with EDTA, dinitrophenol, CdCl2 and different temperature shifts affects significantly less plasmid transformation of protoplasts than plasmid and chromosomal transformation of competent cells. These data point out the different mechanisms of DNA uptake by competent cells and protoplasts.

Bacillus subtilis↗

Effects of chlorhexidine diacetate and cetylpyridinium chloride on whole cells and protoplasts of Saccharomyces cerevisiae.

Chlorhexidine diacetate (CHA) and the quaternary ammonium compound, cetylpyridinium chloride (CPC), were fungicidal to Saccharomyces cerevisiae A364A and CHA to its mannoprotein mutant LB6-5D. Both CHA and CPC induced leakage of K+ and pentose material from both strains and both agents induced protoplast lysis as well as interacting with crude cell sap. Differences were observed between CHA and CPC in protoplast lysis and in cell sap interaction. Q25 values for A364A with CHA as test agent were 5.6 and 1.6 (fungicidal activity, depending on method of calculation), 1.46 (cell sap interaction) and 0.77 (leakage of pentoses). A sub-lytic concentration of CHA reduced considerably protoplast regeneration. Although the plasma membrane is an important target site for both agents, interaction with other cellular components also contributes to fungal death.

Cetylpyridinium↗

[Labeling of yeast protoplasts by neutral red and nile blue for fusion experiments].

Neutral red and Nile blue were found to be suitable for staining protoplasts from various strains of Saccharomyces cerevisiae. The absorption of stains by the protoplasts was dependent on the pH value, for both stains. At a pH of 7.9 the protoplasts of the fusion partners could be stained differently and therefore it was possible to observe the fusion between the various strains of yeast. Consequently the prerequisites were created for the following micromanipulation.

Fluorescent Dyes↗

[Interspecific protoplast fusion between Bacillus thuringensis Bt-3701 and Bacillus megaterium Bm-107].

The results of the interspecific protoplast fusion between B. thuringensis sub. kurstaki Bt-3701 which has pesticide ability, and B. megaterium var. phosphaticum Bm-107 which has decomposing phosphate activity, were reported. High frequency of protoplast formation and regeneration was obtained with 4h activated Bm-107 treated by 100 micrograms/ml lysozyme, and with 2h activated Bt-3701 treated by 3% glycin and mild temperature. Using 40% PEG and 5% nascent Ca2+ to treat the parential protoplast mixture for 3 min at 37 degrees C, 4 stable fusants were obtained. Biological tests show that they have both pesticide ability and decomposing phosphate activity, but which are weaker than that of parential strains.

Bacillus megaterium↗

[Effect of cyclosporin A and amphotericin B on protoplasts of Tolypocladium inflatum].

When added to the suspension of washed cells of Tolypocladium inflatum (cyclosporine-producing organism) in a concentration up to 100 micrograms/ml, cyclosporine A had no effect on permeability of the cell covers as judged by the quantity of the metabolites with the absorption maxima at 260 nm that leaked to the medium. When cyclosporine was added to the suspension of the T-inflatum cell protoplasts there was a significant increase in the metabolites leaking. Impairment of the permeability of the protoplast membranes was more marked in low potent strains. Impairment of the protoplast membrane permeability by amphotericin B was equal in both the high and the low potent strains and in this respect it was more active than cyclosporine.

Amphotericin B↗