Sporulation of Spheroplasts in Saccharomyces cerevisia

Spheroplasts of Saccharomyces cerevisiae sporulated in 0.1 Mpotassium acetate solution which contained 0.8 M sorbitol ormannitol as the osmotic stabilizer. The appearance of matureasci in both spheroplasts and intact cells was retarded by theaddition of the osmotic stabilizer. Sporulation was repressedmarkedly when 0.6 M KCl was used as the osmotic stabilizer inthe sporulation medium. The germination rate of the spores formedin spheroplasts was 97%. Tetrad analysis showed that meiosiswas normal during the sporulation of spheroplasts. (Received September 5, 1980; Accepted November 29, 1980)     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces Cerevisiae

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effects of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is disscussed.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces cerevisiae.

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.     

Regeneration of Spheroplasts in a N2-Fixed Filamentous Blue-Green Alga Anabaena cylindrica

Over 90% of cells of Anabaena cylindrica growing in the medium containing 0. 1 mol/L KC1 for 7～9 d transformed into spheroplasts or semispheroplasts which were either sensitive or not sensitive to hypotonic condition. After treating the materials with 0. 1% lysozyme at 28 ℃ for 3～4 h the transformed spheroplasts were almost 100% sensitive to the hypotonic condition. The spheroplasts then regenerated and divided through culture in the inorganic medium containing 0.15 mol/L CaCl2 with a rate over 25 %. The regeneration of different spheroplasts was not synchronous, the fastest division being after 3 d. Cell division was mainly equational but also irregular division or budding.     

Antigenic structure of Brucella suis spheroplasts

Baughn, Robert E. (University of Tennessee, Memphis), and Bob A. Freeman. Antigenic structure of Brucella suis spheroplasts. J. Bacteriol. 92:1298-1303. 1966.-Immunoelectrophoresis was used to differentiate between the antigenic mosaics of normal cells of Brucella suis and of spheroplasts prepared by treatment with penicillin, glycine, and a combination of these agents. Smooth cells possessed at least 13 antigens, 10 of which were precipitated with homologous antiserum. Three additional antigens were visualized by reaction with spheroplast antisera. Spheroplasts induced with glycine were the least complex, with only six antigens. Penicillin-glycine spheroplasts were similar, but possessed one additional antigen. Penicillin spheroplasts were the most complex, with eight antigens. Although there appeared to be quantitative differences between the antigens of spheroplasts and normal cells, no completely new antigens were detected in spheroplasts. Serum absorption studies indicated that four antigens were associated with the surface of normal B. suis, none of which occurred in spheroplasts.     

Toxin Production in Clostridium botulinum as Demonstrated by Electron Microscopy

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.     

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.     

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.     

Identification ofEscherichia coli spheroplasts by the fluorescent-antibody staining technique

Spheroplasts ofEscherichia coli were produced by penicillin or lysozyme-ethylenediaminetetraacetic acid and examined by the direct fluorescent-antibody staining technique. Most spheroplasts stained with somatic-O fluorescent antibody exhibited brilliant peripheral fluorescence with localized areas of irregular staining. Electron micrographs showed that these spherical structures had considerable amounts of cell wall fragments associated with them. Two strains ofE. coli employed in the present study required different concentrations of penicillin for the conversion of all cells in an exponential culture to spheroplasts. Slight differences in lysozyme sensitivity were also encountered with these strains. The direct fluorescent-antibody staining technique was effective for the rapid identification ofE. coli spheroplasts in mixed cultures.     

Spheroplast fusion and heterokaryon formation in Mucor racemosus

Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to spheroplasts by using commercial chitinase and Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed in a buffered Ca(NO3)2 solution and fusion occurred. After cell wall regeneration, prototrophs were isolated. The frequency of heterokaryon formation was 1.45 X 10(-4). Prototrophic isolates segregated parental nuclei at a high frequency, indicating that heterokaryons had formed.     

Penicillin-Binding Component of Bacillus cereus

(14)C-penicillin is irreversibly bound by Bacillus cereus 569. Incubation of penicillin-treated cells in a cell wall digestion medium results in solubilization of approximately 60% of the irreversibly bound lable. The extent of the solubilization is the same when cells are prepared by either a cold or 37 C treatment procedure. However, spheroplasts prepared by the cold treatment are leaky. When the resulting spheroplasts are incubated in supplemented medium, reduced rates and levels of penicillinase synthesis, relative to induced whole-cell controls, are observed. Spheroplasts from both cold and 37 C prepared cells exhibit this phenomena, although the spheroplasts from 37 C prepared cells synthesized approximately sixfold higher levels of penicillinase. The size distribution of the label solubilized during the preparation of spheroplasts was examined by using Bio Gel P-150 columns. Although no label appeared in the exclusion volume fractions when the cell wall digest of the 37 C treated cells was chromatographed, approximately 10% of the label from cold-treated cells did appear. These results suggest that the presence of irreversibly bound penicillin is required for the synthesis of induced levels of penicillinase and that the irreversibly bound penicillin can be solubilized as a labile complex with material which is excluded from BioGel P-150. It may be concluded that the penicillin-binding lipoprotein complex which has been previously observed is the penicillin-specific binding site. However, the location of this complex in relation to the cell membrane could not be determined.     

In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin

Spheroplasts of the unstable l-form of Proteus mirabilis with fragile, shape defective cell walls grown in medium containing 120 mg/l penicillin G and then killed and permeabilized by ether treatment, were capable of in vitro synthesis of peptidoglycan from the precursors UDP-GlcNAc and UDP-MurNAc-l-Ala-d-Glu(ms-A₂pm-d-Ala-d-Ala). The in vitro peptidoglycan was extensively peptide-crosslinked, indicating a continuing function of peptidoglycan transpeptidase in the spheroplasts. The seven penicillin-binding proteins (PBPs) of P. mirabilis with their functions as multiple peptidoglycan transpeptidases were shown to be saturated in the spheroplasts and thereby functionally inactivated by the penicillin of the growth medium to a very different degree. Complete or almost complete saturation occurred with the PBPs 1A, 1B, and 3, for which functions as indispensible transpeptidases in Escherichia coli have been postulated. In contrast, PBPs 5 and 6 were not saturated in the l-form spheroplasts. Transpeptidase function has been described previously in PBP 5 of P. mirabilis. The working hypothesis is proposed that synthesis of the functionally defective peptidoglycan of l-form spheroplasts in the presence of penicillin takes place with transpeptidase function of PBP 5.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Spheroplast induction inAnabaena variabilis Kütz andA. azollae stras

Summary Spheroplasts were obtained by lysozyme treatment of 48 hour (4– 8cells) akinete germlings of the cultured cyanobacteriaAnabaena variabilis andA. azollae originally isolated from the leaf cavity of the fernAzolla pinnata. The osmotic stabilizer was 0.5 M sucrose. At least 50% of the cells in a short filament became spheroplasts after 1–4 hours in lysozyme (1 mg/ml) in incubation medium at 34 °C, with greater than 75% viability after 2 hours. The spheroplasts were osmotically fragile and showed intense chlorophyll autofluorescence in UV light. In phase microscopy, treated cells appeared larger, became spherical and lost some of their optical refraction. Transmission electron microscopy confirmed the loss of the peptidoglycan layer and the partial remains of the outer membrane after lysozyme exposure. We previously obtained protoplasts ofAzolla fern leaf cells so that we now can study the recognition sites in both members of theAzolla/Anabaena nitrogen fixing symbiosis during cell wall degradation and regeneration.     

Obtaining spheroplasts from the agents of glanders and melioidosis and separation of membrane structures from them]

Spheroplasts were obtained from the causative agents of glanders and melioidosis under the effect of lysozyme and antibiotics. In the capacity of an inducing agent lysozyme was effective in high concentration only (0.4%); preliminary washing and incubation in sucrose were necessary to obtain glanders spheroplasts. Of the antibiotics studied penicillin was more useful for obtaining melioidosis spheroplasts and ampicillin--for glanders spheroplasts. Membrane preparations were derived from the spheroplasts of glanders and melioidosis causative agents.     

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.     

Multiplication of Bacteriophage P22 in Penicillin-Induced Spheroplasts of Salmonella typhimurium

The spheroplasts of Salmonella typhimurium (LT2) prepared by treatment with penicillin were capable of adsorbing phage P22 C(1). The normal multiplication of the phage took place, although the burst size was reduced to one-fourth of that in intact cells. Rate of incorporation of (14)C-thymidine into spheroplasts was increased severalfold on phage infection. Multiplication of C(+) also took place, but no lysogeny could be established in spheroplasts. Furthermore, spheroplasts prepared from cells lysogenized with wild-type phage, LT2 (C(+)), and a temperature-inducible C(2) mutant, LT2(tsC(2)), were not inducible. Unlike normal cells, both mitomycin C and actinomycin D interfered with the phage multiplication in spheroplasts. The spheroplast system offers great advantages in the study of the synthesis of nucleic acids and proteins in phage-infected LT2.     

硅藻土在青霉素G酰化酶提纯中的应用

国产硅藻土经氢氧化铵处理后可用于从发酰液中直接取青霉素Ｇ酰化酶,平均吸附量为９０Ｕ／ｇ。吸附的酶可用２２％硫酸铵－０．３ｍｏｌ／ＬＰＢＳ（ｐＨ８．０）溶液洗脱。平均比１８Ｕ／１８Ｕｍｇ蛋白（ＮＩＰＡＢ法）。硅藻土可反复使用。苯乙酰胺－Ｓｅｐｈａｒｏｓｅ４Ｂ树脂可对酶作进一步的纯化。     

Metabolism of [14C]glucose by regenerating spheroplasts of Candida albicans

Spheroplasts of Candida albicans were regenerated in [14C]glucose and buffered magnesium sulphate (0.1 M-Tris/HCl; 0.5 M-MgSO4, pH 7.2) at 35 degrees C. Uptake of glucose by spheroplasts was faster than that by intact yeast cells. After 6 h, 65% of the glucose taken up by the yeast appeared as CO2 and 30% was incorporated into the cellular material. With spheroplasts, 55% of the glucose taken up was expired as CO2, 25% was excreted into the medium as other metabolites and 20% was incorporated into the cells. The regenerating spheroplasts excreted 14C-labelled carbohydrates into the medium which were fractionated on a Sephadex G-15 column. Acid hydrolysis of the low molecular-weight fraction yielded the following sugars: mannose (75.7%), fucose (3.8%), arabinose (3%), galactose (2.1%) and an unidentified monosaccharide (14%). Spheroplasts did not incorporate mannoprotein into the regenerated wall. The wall carbohydrate from regenerated spheroplasts was fractionated on the basis of solubility in sodium hydroxide. The alkali-insoluble fraction was analysed by sequential enzyme hydrolysis; 40% of the incorporated counts were associated with beta (1----3)-linked glucan and 50% with a mixed glucan comprising beta (1----3)- and beta (1----6)-linkages and chitin.     

Natural penicillin resistance of Francisella tularensis

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.