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PubMed1982

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.

Cell Division↗
PubMed1976

An ultrastructural analysis of protoplast-spheroplast induction in Cryptococcus neoformans.

Protoplasts-spheroplasts of a human isolate of Cryptococcus neoformans were prepared using the gut enzyme of Helix pomatia. The induction process, as studied by transmission electron microscopy, occurred in two stages. Early in the induction process, protoplasts-spheroplasts emerged from whole cells through a break in the cell wall-capsule envelope. Later, a gradual dissolution of the entire cell wall occurred releasing the intact protoplast-spheroplast. A comparison of protoplasts-spheroplasts with normal untreated cells revealed that the degree of cellular vacuolation as well as the resolution of cellular organelles was similar.

Cell Wall↗
PubMed1983

An improved method for the preparation of mycobacterial spheroplasts and the mechanism involved in the reversion to bacillary form: electron microscopic and physiological study.

An efficient method is described for preparing spheroplasts and protoplasts by treating bacillary cells of Mycobacterium smegmatis with precise concentrations of L-glycine (followed by lysozyme). This improved procedure was widely applicable to many rapidly growing mycobacteria by selecting the concentrations of glycine suitable for the individual strains used. The process of reversion of spheroplasts to original bacillary form on solid and in liquid media, as revealed by electron microscopy, appeared to involve the formation of an internal elementary or initial body with subsequent budding from the spheroplast. The internal membrane systems appeared to function in the induction of initial bodies and in the maturation of elementary bodies to become dividing forms. Possible mechanisms involved in the development of bacilli from spheroplasts are discussed.

Bacteriological Techniques↗
PubMed1976

The pathogenicity of spheroplasts of Mycobacterium tuberculosis.

Guinea pigs were injected with spheroplasts of Mycobacterium tuberculosis and were sacrificed after various intervals to study the state of the organisms and to evaluate the extent of disease. The spheroplasts persisted in the body of the animals for a considerable length of time. Their presence, however, was not associated with either the development of tuberculin hypersensitivity or pathologic changes. It was found that the spheroplasts were pathogenic only when reverted in the animals to bacterial forms. The reversion of the spheroplasts to bacillary state and the subsequent initiation of infection were dependent on the duration of their stay within the host.

Animals↗
PubMedDate not supplied

Phospholipids and glycerides composition during spheroplasts formation of Mycobacterium smegmatis ATCC 14468.

The phospholipids and glycerides composition of spheroplasts of Mycobacterium smegmatis ATCC 14468 was examined. The percent total phospholipids in total lipids as well as cardiolipin were found to be higher in spheroplasts as compared to their parent forms. Increase in cardiolipin and free fatty acids content and decrease in triglycerides levels were observed during spheroplasts formation. The results suggest that increase in cardiolipin content in spheroplasts is an adaptational change concomitant with the loss of cell walls.

Carbon Radioisotopes↗
PubMed2000

Cystamine transport in spheroplasts of Saccharomyces cerevisiae.

This work is the first demonstration that cystamine is actively accumulated in spheroplasts of Saccharomyces cerevisiae. We have identified and quantitatively determined the transported cystamine in extracts of spheroplasts that have been incubated over different time periods and in the presence of different amounts of cystamine. The method used, already reported in literature for the identification of natural aliphatic polyamines in biological fluids, consists of a derivatization of spheroplast extracts with dabsyl-chloride and subsequent chromatographic analysis in HPLC. Our results show that cystamine accumulation is a function of time, it increases up to 2.5 min then decreases. Transport is inhibited by natural aliphatic polyamines, which, at the same concentration of cystamine (1 mM), cause a decrease in cystamine transport of about 90% for spermidine, 50% for spermine and only 15% for putrescine. Furthermore, transport is energy-dependent as demonstrated by a significant decrease observed in the presence of 2,4-dinitrophenol, ouabain and vanadate. In particular 0.2 mM ouabain causes a decrease of more than 60% in cystamine transport. Our data suggest that cystamine is transported in Saccharomyces cerevisiae spheroplasts via the same polyamine transport system(s) known to be operating in higher eukaryotic cells.

Adenosine Triphosphatases↗
PubMed1991

Investigation of osmotically stable spheroplasts from two strains of Escherichia coli ML-35, W7-M5.

Osmotically stable spheroplasts were produced from Escherichia coli ML-35 and W7-M5 using either 1 min exposure to polymyxin B or 10 min exposure to Tris/EDTA, followed by 1 to 3 h incubation with lysozyme. Spheroplast membrane permeability studies were conducted using paired radioactive probes with E. coli ML-35. Experiments with 14C-sucrose-16 kD 3H-dextran indicated that the outer membrane had lost its barrier to 16 kD dextran. Parallel experiments with 81 kD 3H-dextran indicated that the outer membrane was impermeable to the larger dextran. EDTA treated cells also showed outer membrane permeability to 16 kD dextran. Cytoplasmic membrane integrity was confirmed using 14C-sucrose and 3H2O before and after exposure to polymyxin B and EDTA. Scanning electron microscopy showed that a rough surface on polymyxin B produced spheroplasts while Tris/EDTA spheroplasts showed the same smooth surface as control cells.

Cell Membrane↗
PubMedDate not supplied

[Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization].

Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.

Ammonia-Lyases↗
PubMedDate not supplied

Growth and macromolecular synthesis of spheroplasts of Mycobacterium smegmatis ATCC 14468.

The growth and synthesis of macromolecules in spheroplasts of Mycobacterium smegmatis ATCC 14468 were found to be in a balanced state. Spheroplasts were not lysed during preparation and subsequent incubation, and were not contaminated with whole cells. Limited growth of spheroplasts was not due to depletion of medium constituents. High molar concentrations of osmotic stabilizer did not inhibit balanced growth. These studies establish that spheroplasts are metabolically active and can be used as a tool in cell wall synthesis and in genetic studies.

Bacteriological Techniques↗
PubMed1976

[Various data concerning the ultrastructure of Proteus spheroplasts].

Spheroplasts were obtained under the action of penicillin on the same medium which was later used to obtain the L-forms. Spheroplast formation started 15 to 20 minutes after the addition of penicillin and reached the maximum in 2 hours. The first dividing forms were revealed at that time, and this division continued for at least 24 hours. The majority of the cells represented spheroplasts surrounded by outer and cytoplasmic membrane, and only some--true protoplasts--had cytoplasmic membrane alone. Division was anomalous in comparison with the bacterial forms with the cell wall: it was noted that either both daughter cells were surrounded by a common outer membrane, or one daughter cell had two membranes serving as a spheroplast and the other--one membrane, serving as a true protoplast. Individual vesicles and myelin-like structures were found to be extruded into the periplasmic space or directly into the surrounding environment. In the latter case pearl-like structures described by Ryter in the formation of protoplasts in bacilli were observed. However, in the Proteus such structures were largely formed by the material of the plastic layer of the cell wall, and to a lesser extent--by the lipoproteid membrane.

Cell Division↗
PubMedDate not supplied

[Comparative study of the functions of the spheroplasts and cells of the blue-green alga Anabaena variabilis].

The effect of light was studied with the spheroplasts of the blue-green alga Anabaena variabilis. Contrary to the intact cells, the spheroplasts did not synthesize nucleic acids and pigments in the light. These components of the spheroplasts were decomposed in the light, and the remaining chlorophyll was incapable of luminescence. The rate of oxygen uptake increased upon the incubation of the spheroplasts in the light. Changes of the functions induced by lysozyme in the cells of A. variabilis are irreversible, contrary to those which are caused by the incubation in the darkness and can be restored if the cells are transferred into the light.

Chlorophyll↗
PubMed2000

Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response.

The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the sigmaE stress response. Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Amino Acid Sequence↗
PubMed1991

[Spheroplast formation in clinical isolates of gram-negative bacteria by beta-lactam antibiotic in the presence of Ca2+ or Mg2+].

Induction to cell wall-deficient bacteria has been suggested to be a cause of intractable and opportunistic infection after chemotherapy. Spheroplast formation by beta-lactam antibiotic in not so high osmotic pressure was investigated in six species of gram-negative bacteria. Some species of gram-negative bacteria were induced to form spheroplast at a high rate by 1:10 concentration of ceftizoxime in the presence of Ca2+ or Mg2+. Especially in 38% of Proteus mirabilis and P. vulgaris, more than 40% of the original cells were induced to form spheroplast by ceftizoxime in a medium supplemented with 40 mM Ca2+. The same formation rate was also found in 22% of Serratia marcescens. Formation rates in the drug sensitive strains of S. marcescens were enhanced as the drug concentration increased. Ca2+ was more effective in spheroplast formation than Mg2+.

Calcium↗
PubMed1969

Inducible synthesis of beta-galactosidase in disrupted spheroplast of Escherichia coli.

A membrane preparation obtained from osmotic lysate of spheroplasts of Escherichia coli cells showed an activity of synthesizing beta-galactosidase which was dependent upon oxidative phosphorylation. The synthesis was inhibited by the addition of actinomycin D or of chloramphenicol. The beta-galactosidase synthesized in the membrane preparation was completely released into the medium, while that synthesized in the spheroplasts and intact cells remained within the cells. The minimum concentration of the inducer, methyl-beta-d-thiogalactoside, required for the induction of beta-galactosidase was 5 x 10(-5)m for intact cells, 3 x 10(-4)m for spheroplasts and 1 x 10(-3)m for membrane preparation. Incorporation of labeled glucose into insoluble components in membrane preparation was extremely low compared with that in intact cells or in spheroplasts. Based on these and other observations, the nature of this membrane preparation is discussed in relation to the structure of E. coli cells.

Bacteriolysis↗
PubMed1970

Stabilizing effect of colicin E2 on the "spheroplast membrane".

Cells of Escherichia coli W3110 and its thymineless mutant, both of which are colicin E2 sensitive, were treated with colicin E2, and then converted to spheroplasts. These spheroplasts seemed to be more stable than those from untreated cells; suspensions of spheroplasts of untreated cells were lysed spontaneously and the turbidity was reduced by approximately 45% on incubation with ethylenediaminetetraacetic acid-lysozyme, whereas suspensions of spheroplasts of colicin E2-treated cells showed 25% reduction in turbidity. This change was irreversible and 5 min of treatment with colicin E2 at 37 C was necessary for stabilization. This process was inhibited by 2,4-dinitrophenol or streptomycin. Cells harboring the colicin E2 factor were not affected by treatment in this way with colicin E2. Alteration of composition of phospholipids was not observed.

Cell Membrane↗
PubMed1966

Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli.

Hirokawa, Hideo (The University of Tokyo, Tokyo, Japan). Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli. J. Bacteriol. 91:125-128. 1966.-Spheroplasts of Escherichia coli B-054 were formed by penicillin, and the properties of rod cells reversed from the spheroplasts were examined. It was found that the rod cells were not capable of forming colonies when placed in a minimal medium immediately after the reversion, the postaddition of the nutrient to this medium being without effect. In this respect, the so-called residual cells which did not convert to spheroplasts by penicillin treatment behaved just as the reversed rod cells. These two types of cells, the reversed and residual rod cells, however, could grow up to form colonies when they were cultivated in a complex medium immediately after the reversion. The nutrient requirement and penicillin sensitivity of the progeny of these cells were found to be quite similar to those of the original untreated cells. Accordingly, it was concluded that the nutritional defect induced by penicillin treatment was not a hereditary change.

Escherichia coli↗
PubMed1966

Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts.

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts. J. Bacteriol. 91:595-600. 1966.-The effect of streptolysin S, the group D hemolysin, and phospholipase C (the alpha toxin of Clostridium perfringens) on whole cells and spheroplasts or protoplasts of three strains of streptococci and Micrococcus lysodeikticus was tested. Viability, C(14)-glycine uptake, and lysis were measured. The group D hemolysin and phospholipase C were active against whole bacteria; streptolysin S was not. All three substances were active on spheroplasts. A partially resistant mutant derived from a strain sensitive to the group D hemolysin was also partially resistant to streptolysin S and phospholipase C. Antimycin A protected spheroplasts from streptolysin S but not from the group D hemolysin.

Antimycin A↗
PubMed1969

Electron microscopy and viability of lysostaphin-induced staphylococcal spheroplasts, protoplast-like bodies, and protoplasts.

The cell walls of a selected isolate of Staphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal "protoplasts" reported by other investigators and should serve as the basis for a variety of fundamental investigations.

Anti-Bacterial Agents↗