Growth Of Protoplasts Research Articles

Proteinkristalle und Tubuli-Bündel in Hefezellen: 1. Assemblierung und Desassemblierung

Protein crystals and bundles of tubules (tb) are induced inside of intact cells of Saccharomyces carlsbergensis and S. cerevisiae by incubation in hypertonic media both at room temperature and at 0 °C within a few minutes or by preparation of protoplasts. The induction is not possible in cells of Kluyveromyces fragilis and Rhodosporidium diabovatum. Formation of these structures depends on osmolality of the hypertonic media not on the kind of substances used. In cells previously cultured in medium containing glucose (5 %) instead of lactate (2 %) or ethanol (1 %) these structures can be induced only in few cases. Crystals and tb disappear within 1—4 hours provided that osmotic shock is followed by growth of cells or protoplasts.

Naturally occurring differential drug sensitivities of cultured plant protoplasts

The growth of protoplasts isolated from petunia and tobacco leaves has been assessed in the presence of a range of concentrations of various amino acid analogues, growth substances and other drugs. Some of the compounds showed no differential effect on the growth of the protoplasts. Several of the drugs, however, showed a pronounced differential effect preventing the growth of tobacco protoplasts while at the same concentration allowing the growth of petunia protoplasts, and with certain other drugs the converse situation prevailed. It is suggested that such naturally occurring differential drug sensitivities might provide a basis for the complementation selection of somatic hybrids arising from the culture of fused protoplasts.

The induction of aggregation and fusion of Dancus carota protoplasts by polyethylene glycol

Summary A method for induction of high aggregation and fusion frequency is described. About 10% dinucleated fused protoplast were obtained if Daucus carota protoplasts from a normally dividing cell suspension in protoplast growth medium were treated in a final concentration of 28% (w/w) polyethylene glycol at 25°C. The influence of salt composition and concentration on protoplast aggregation and fusion was studied as well as effects of different temperatures, pH and protoplast age and condition. Light- and electron micrographs show the strong adhesion and fusion of cell membranes and the osmotic changes in the protoplasts induced by polytehylene glycol.

Inhibitory effect of 2‐deoxy‐glucose on cell wall synthesis in cells and protoplasts of Schizosaccharomyces pombe

AbstractA study has been made of changes in cell walls and ultrastructure of cells and regenerating protoplasts of Schizosaccharomyces pombe grown in different concentrations of 2‐deoxyglucose (2DG). At concentrations of 0.1% and higher the inhibitor reversibly blocked growth of cells and protoplasts and completely inhibited cell wall synthesis. Concentrations of 0.05% and lower allowed protoplasts to grow and induced early or late lysis in normal cells. On the surface of protoplasts only the fibrillar component was formed; secretion of the soluble fraction ceased. The early lysis of the cells was a result of degradation of cell wall material at growing sites (concentration of 2DG 0.02–0.05%), the late lysis occurred in cells with weakened growing regions, where aberrant material was deposited (concentrations 0.01–0.02%). Concentrations of 2DG below 0.01% did not affect the growth of the cells or the reversion of protoplasts to normal cells. 2DG was not incorporated into the walls of cells or regenerating protoplasts, and did not influence the ultrastructure of cells or protoplasts except for the effects on the cell wall.

Inhibitory effect of 2-deoxy-glucose on cell wall synthesis in cells and protoplasts of Schizosaccharomyces pombe.

A study has been made of changes in cell walls and ultrastructure of cells and regenerating protoplasts of Schizosaccharomyces pombe grown in different concentrations of 2-deoxyglucose (2DG). At concentrations of 0.1% and higher the inhibitor reversibly blocked growth of cells and protoplasts and completely inhibited cell wall synthesis. Concentrations of 0.05% and lower allowed protoplasts to grow and induced early or late lysis in normal cells. On the surface of protoplasts only the fibrillar component was formed; secretion of the soluble fraction ceased. The early lysis of the cells was a result of degradation of cell wall material at growing sites (concentration of 2DG 0.02–0.05%), the late lysis occurred in cells with weakened growing regions, where aberrant material was deposited (concentrations 0.01–0.02%). Concentrations of 2DG below 0.01% did not affect the growth of the cells or the reversion of protoplasts to normal cells. 2DG was not incorporated into the walls of cells or regenerating protoplasts, and did not influence the ultrastructure of cells or protoplasts except for the effects on the cell wall.

Growth of Streptococcal Protoplasts and L-Colonies on Membrane Filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.

Formation, growth, and regeneration of protoplasts of the green alga,Uronema gigas

Separate protoplasts were obtained by the action of snail gut juice enzymes on the cell walls of the green algaUronema gigas. The cultivation of the protoplasts in mineral media caused only their enormous growth; in the presence of glucose a fibrillar network was formed on the surfaces of the growing protoplasts. Only after the addition of pectin the regeneration of the cell wall and the renewal of their morphogenesis could be observed.

Studies on the mode of action of novobiocin

Novobiocin (NB) reduced lysis and prevented spheroplast formation in penicillin-treated E. coli. It caused a slow lysis of preformed spheroplasts when these were suspended in an environment conducive to their growth, and prevented reversion of spheroplasts to rods. It did not lyse “resting” protoplasts of Bacillus megaterium, but inhibited the growth of protoplasts, and brought about slow lysis of these forms.

Auxin‐Induced Expansion Growth of Cells and Protoplasts of Yeast

AbstractUsing an auxin‐responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts. Indole‐3‐acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred. The auxin‐induced expansion growth was inhibited by an antiauxin, trans‐cinnamic acid. Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin‐induced cell expansion growth. Protoplasts did not expand in response to auxin under the condition where intact cells did. The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion. High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

Evidence for excretion of certain enzymes during growth of protoplasts of Bacillus megaterium

Evidence for excretion of certain enzymes during growth of protoplasts of Bacillus megaterium

Growth and division of protoplasts of Bacillus megaterium and inhibition of division by penicillin.

Protoplasts of Bacillus megaterium grew well and divided in nutrient broth containing 0.5 m NaCl as the stabilizer. Protoplasts also grew when sucrose or succinate was used instead of NaCl; however, no division phenomena were observed. The sequence of growth and division was similar to the results obtained by McQuillen. Protoplasts enlarged from 1.8 to 3.5 mu in diameter, and then a small protuberance formed in the enlarged cells. The nodules enlarged until eventually symmetrical dumbbell-shaped bodies were formed, which then separated into two daughter protoplasts having a diameter of about 2.5 mu. Deoxyribonucleic acid and ribonucleic acid were halved during division. Penicillin inhibited the division of protoplasts, though the growth was not influenced by the drug. Membrane-bound amino sugar content was considerably reduced when the cells were grown in the presence of penicillin. These results suggest that organized murein formed on the protoplasts membrane may play an important role in the septation process.

DNA and protein synthesis in the growing yeast protoplast

The growth of protoplasts of three strains of brewing yeasts in the gelatinsolidified media, comprising malt extract, brewers' wort and chemically defined types gave no evidence even after 7 days of cell wall regeneration and division by budding. Estimates were made of the content of deoxyribonucleic acid (DNA) and of protein in protoplasts of the three strains, and the changes in content with time over 12 hr in liquid malt extract medium using two of them. The DNA content of the protoplasts approximately doubled in about 6 hr after which time the rate of synthesis fell almost to zero. Protein synthesis continued for about 10 hr before the rate declined. The rate of synthesis was substantially less than in young whole cells under similar conditions. In both cells and protoplasts the pattern of protein synthesis was not logarithmic. This was probably due to the unstirred culture conditions. There is evidently no direct relationship between protein and DNA content in protoplasts. The failure of the protoplasts to bud and form new cells evidently is not due to a failure to synthesize DNA and other possible explanations are discussed.

Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium.

Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium. J. Bacteriol. 87:1044-1050. 1964.-Penicillin and cycloserine do not inhibit the growth of protoplasts of Bacillus megaterium, indicating that inhibition of cell-wall synthesis is the only significant process by which they inhibit growth of bacteria. In contrast, bacitracin and vancomycin inhibit growth of protoplasts and bacteria at similar concentrations, indicating that they have important sites of action other than their known inhibition of cell-wall synthesis. At concentrations which inhibit mucopeptide synthesis, penicillin, bacitracin, and vancomycin each cause an increased rate of efflux of K ions from growing bacteria. This effect of penicillin is prevented by chloramphenicol or hypertonic sucrose, whereas the effects of bacitracin and vancomycin are unchanged under these conditions. It is concluded that bacitracin and vancomycin have direct effects on the cytoplasmic membrane, and it is proposed that their inhibition of cell-wall synthesis could be a consequence of these effects. Bacitracin and vancomycin do not compete with penicillin for binding to cells of B. megaterium, a further indication that they have a different primary site of action.

Inhibition by antibiotics of the growth of bacterial and yeast protoplasts.

Shockman, Gerald D. (Temple University School of Medicine, Philadelphia, Pa.) and J. Oliver Lampen. Inhibition by antibiotics of the growth of bacterial and yeast protoplasts. J. Bacteriol. 84:508-512. 1962.-The characteristics and requirements for growth of bacterial (Streptococcus faecalis) and yeast (Saccharomyces cerevisiae) protoplasts were established and the effect of a variety of antibacterial and antifungal antibiotics determined. A clear differentiation was obtained between such inhibitors of bacterial cell wall synthesis as penicillin and cycloserine, which did not prevent protoplast growth, and all others, antibacterial and antifungal, which inhibited protoplasts and intact organisms at the same range of concentration. Novobiocin, previously reported to inhibit bacterial wall synthesis, was also effective against a reaction(s) essential to the growth of S. faecalis protoplasts. The antibacterial action of streptomycin, neomycin, and kanamycin was essentially eliminated by the high salt concentration needed to maintain the protoplasts. Removal of the cell wall did not significantly increase antibiotic susceptibility of a resistant species. Protoplasts of Bacillus megaterium were insensitive to the antifungal agent, nystatin, and did not bind it to any detectable degree. Thus, the yeast or bacterial cell wall does not appear to play a major role in determining relative antibiotic susceptibility by masking internal sensitive target sites. A variety of antifungal antibiotics tested on the growth of log-phase yeast cells failed to produce osmotically fragile forms.

Physical conditions as important factors for the regeneration of naked yeast protoplasts.

IN our previous papers1 we have discussed the growth and regeneration of naked yeast protoplasts of Saccharomyces cerevisiae obtained by means of cell wall autolysis. In the former experiments the growth and regeneration took place on solid synthetic media. The frequency of regeneration was low on these media; while nearly all the protoplasts were capable of growth, in fact only about 1 per cent regenerated into new yeast cells (in a complicated manner formerly described).

The growth and division of bacterial protoplasts

The mode of release, growth, and division of naked bacterial protoplasts is described. The significance of the observations made upon these structures is discussed and some possible implications are referred to.

Cytological and chemical studies of the growth of protoplasts of Bacillus megaterium.

Aeration of protoplasts of Bacillus megaterium in a succinate buffered nutrient broth led to marked growth similar to that already described by McQuillen, and the degree of chromatin synthesis in these growing forms prompted a combined cytological and chemical study. Growth was followed by phase contrast and by Feulgen stains, as well as by lipide phosphorus, nucleic acid, and protein analyses. In slide cultures, growth and compression led to monstrous flattened forms with readily visible, but coalescent nuclear structures. In fluid cultures, the protoplasts grew as phase dense spheres. Orderly reproduction of apparently discrete nuclear bodies was observed during the initial hours of spherical growth, but in older cultures, the chromatin arrangement tended to be more haphazard and was influenced by the concentration of Mg ions. In the same medium, protoplasts free of lysis showed a linear rise in optical density, while vegetative cells exhibited an exponential increase. However, protoplasts were able to synthesize DNA at the same rate as vegetative cells, but their increase of RNA was always less. Thus, as they grew, the ratio RNA/DNA fell. The lipide P increased in proportion to the expanding surface. With growth and lysis, large amounts of water-insoluble slime accumulated. Analyses indicate it to be a phospholipoprotein material containing some RNA.

Growth and Properties of Bacterial Protoplasts

PREVIOUS papers on the subject of bacterial protoplasts prepared by the action of lysozyme indicate that the authors have been unable to observe growth and division of these structures over a prolonged period1–3, although indications of activity in this direction have been obtained4,5.

Bacterial protoplasts: growth and division of protoplasts of Bacillus megaterium

Bacterial protoplasts: growth and division of protoplasts of Bacillus megaterium