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PubMed1974

Chemical composition and ultrastructure of native and reaggregated membranes from protoplasts of Bacillus cereus.

Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of Bacillus cereus T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C(15) and C(17) and of normal C(16) acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl(2). The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl(2) concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl(2) concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments.

Bacillus cereus↗
PubMed1975

Protoplast formation and leakage of intramembrane cell components: induction by the competence activator substance of pneumococci.

Treatment of pneumococci with activator (a protein that induces bacterial "competence" to absorb deoxyribonucleic acid molecules and undergo genetic transformation) can cause either protoplast formation or leakage of intracellular components to the medium depending on postincubation conditions. The leaked intracellular components include nucleoside phosphates, beta-galactosidase, deoxyribonuclease, autolysin, and hemolysin. Leakage and protoplast formation are induced by the electrophoretically pure activator, and these phenomena require the same conditions as induction of competence for genetic transformation, namely, genetic capacity for competence, protein synthesis, incorporation of choline, and the optimal pH for activation. It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space. The activity of these autolysin molecules from within would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.

Bacterial Proteins↗
PubMed1975

Degradation of phospholipid and release of diglyceride-rich membrane vesicles during protoplast formation in certain gram-positive bacteria.

Membrane phospholipid was found to be hydrolyzed presumably by an intracellular phospholipase C, and diglyceride-rich membrane vesicles were released from the cells during protoplast formation in Bacillus cereus Bacillus subtilis, Micrococcus lysodeikticus, and Staphylococcus aureus. The released membranes consisted mainly of small vesicles of 50 to 100 nm in diameter. They have a lower density than that of protoplast membranes in all the bacteria tested in the present study.

Acetates↗
PubMed1975

Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae.

The localization of acid phosphatase (EC 3.1.3.2) in secreting protoplasts prepared from Saccharomyces cerevisiae is reported for the first time. Using a Gomori technique we were able to show acid phosphatase at those organelles in the protoplasts which are generally involved in the processes of biosynthesis and secretion of glycoproteins in eukaryotic cells.

Acid Phosphatase↗
PubMed1975

Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis.

Protoplasts (autoplasts) of Streptococcus faecalis were produced by the action of native autolytic N-acetylmuramidase in the absence of added peptidoglycan hydrolases and were grown in osmotically stabilized medium containing L-[3H]lysine and D-[14C]alanine. To reduce the level of muralytic hydrolysis of glycan chains during growth, heat-inactivated cell walls were added to the medium to bind autolytic enzyme, and tetracycline (1 mug/ml) was added to inhibit further enzyme synthesis. Under these conditions, protoplasts synthesized newly labeled peptidoglycan in the form of soluble, infrequently peptide cross-linked glycan chains which were released into the supernatant medium. These relatively large glycan chains were not transferred to exogenously added cell walls.

Alanine↗
PubMed1975

Ultrastructural study of the reversion of protoplasts of Bacillus licheniformis to bacilli.

The reversion of protoplasts of Bacillus licheniformis 6346 His- on a medium containing 2.5% agar has been studied in sectioned material after reaction with a ferritin-conjugated antibody specific to the peptidoglycan isolated from the walls of the bacilli. Freeze etching has also been used. Fibrils of material reacting with the antibody have been detected emerging from isolated areas of the protoplasts after 3 h of incubation. This material gradually covers the cell and can eventually (at 6 h) be seen in freeze-etched preparations as a fringe of up to 400 nm around the cells and covering the surfaces with particles that can be removed by lysozyme. At later stages the wall begins to take on a compact, well-defined appearance that can be seen in sections; however, the cells are still grossly deformed. A transitory emergence, beyond the wall of long fibers of 6 nm in diameter, takes place after about 12 h of incubation. These fibers react with the conjugated antibody and after freeze etching show a regular banded structure. They are probably indentical with the fibers isolated elsewhere (Elliott et al., 1975) and shown to contain all the wall constituents (i.e., peptidoglycan, teichoic acid, and teichuronic acid). These fibers are not detectable in the final stages of reversion.

Antibodies, Bacterial↗
PubMed1979

Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790).

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.

Enterococcus faecalis↗
PubMed1979

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.

Genotype↗
PubMed1982

Thiamine transport in Saccharomyces cerevisiae protoplasts.

Thiamine was found to be accumulated in protoplasts of Saccharomyces cerevisiae in the same manner as in intact cells, suggesting that a soluble thiamine-binding protein in periplasm may not be an essential component of the thiamine transport system of S. cerevisiae. It was also found that thiamine pyrophosphate cannot be taken up by yeast protoplasts.

Acid Phosphatase↗
PubMed1982

Cytoplasmic transfer of oligomycin resistance during protoplast fusion of Saccharomycopsis lipolytica.

Mitotic segregation of oligomycin resistance and oligomycin sensitivity was observed among the prototrophic progeny of protoplast fusion between drug-resistant and drug-sensitive complementary auxotrophs of Saccharomycopsis lipolytica. The transfer of oligomycin resistance by protoplast fusion without karyogamy suggests a cytoplasmic inheritance of this drug resistance determinant.

Ascomycota↗
PubMed1983

Computer-assisted chromosome mapping by protoplast fusion in Staphylococcus aureus.

Protoplasts of genetically marked derivatives of Staphylococcus aureus NCTC 8325 were fused with polyethylene glycol and regenerated without selection. Recombinants possessing one specific resistance marker from each parent were selected from the regenerated population and scored for seven or eight unselected markers. The results of these 9- and 10-factor crosses were entered directly into a programmed microcomputer from prescored replica plates. The data then were condensed into an array of phenotypes, together with the frequency with which each occurred. Further analyses by computer included the calculation of coinheritance frequencies for all possible pairs of markers; after entering a proposed order for the markers being analyzed, the minimum number of crossover events required to generate each phenotypic class was calculated. The linkage relationships of markers, based on the protoplast fusion data, were entirely consistent with the linkage relationships of markers already known to exist within each of the three linkage groups previously defined by transformation. The fusion data defined an arrangement of the three linkage groups into a circular chromosome map and predicted the approximate location of four previously unmapped markers (tet-3490, fus-149, purC193::Tn551, and omega [Chr::Tn551]42) on this map.

Chromosome Mapping↗
PubMed1986

Plasmid instability in regenerating protoplasts of Staphylococcus aureus is caused by aberrant cell division.

Elimination of plasmids from regenerating S. aureus protoplasts occurred when the regeneration medium contained sucrose but not when it contained sodium succinate. This difference was caused by the occurrence of cell division prior to regeneration of the cell wall on sucrose but not on succinate. Coexisting compatible plasmids were cured independently; coexisting incompatible plasmids were cured jointly. These results support the hypothesis that plasmid pools exist as physically sequestered units in protoplasts and that curing is a consequence of the segregation of such units during abnormal division of wall-less organisms.

Cell Division↗
PubMed1986

Genetic analysis of Bacillus stearothermophilus by protoplast fusion.

Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, three-, and four-factor crosses. The order of the markers was hom-1-thr-1-his-1-(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacillus subtilis chromosome. No analogous pur-1 marker has been reported in B. subtilis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.

Chromosome Mapping↗
PubMed1963

Protoplast membrane of Streptococcus faecalis.

Shockman, Gerald D. (Temple University School of Medicine, Philadelphia, Pa.), Joseph J. Kolb, Bohdan Bakay, Margaret J. Conover, and Gerrit Toennies. Protoplast membrane of Streptococcus faecalis. J. Bacteriol. 85:168-176. 1963.-The membrane fraction of Streptococcus faecalis (ATCC 9790) was isolated and purified, by a variety of procedures, from cultures that were grown under closely controlled conditions of physiological age and nutrition. The most satisfactory method required the use of lysozyme-to-cell ratios below 0.01 and the intermediate formation of protoplasts in osmotically protective media. Amino acid analyses of three of the membrane preparations indicated a characteristic and constant, but not unusual, pattern; 42% of the membranes from threonine-depleted and 49 to 55% of the membranes from log-phase cultures were accounted for as protein. Significant quantities of d-alanine or d-aspartic acid were not detected, indicating the absence of contaminating cell-wall substance. Essentially, all of the nitrogen was accounted for as amino acids. The lipid content of membranes from stationary-phase threonine-depleted (36%) and valine-depleted (40%) cultures was significantly higher than the corresponding fraction of exponential-phase cultures (28%). The phosphorus content of the membrane lipid was relatively constant (2.8 to 3.0%), and the nitrogen content was extremely low (0.12 to 0.26%). Thus, changes in the composition of the membrane fraction occurred during the transition of log-phase cells into threonine- or valine-depleted cells.

Alanine↗
PubMed1968

Surface structure of yeast protoplasts.

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.

Cell Membrane↗
PubMed1969

Bacteria-shaped gymnoplasts (protoplasts) of Bacillus subtilis.

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.

Bacillus subtilis↗
PubMed1993

The cowpea mosaic virus M RNA-encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts.

Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.

Fabaceae↗