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PubMed1964

ENZYMES OF THE PYRIMIDINE PATHWAY IN ESCHERICHIA COLI. I. SYNTHESIS BY CELLS AND SPHEROPLASTS.

Taylor, W. Herman (Portland State College, Portland, Ore.), and G. David Novelli. Enzymes of the pyrimidine pathway in Escherichia coli. I. Synthesis by cells and spheroplasts. J. Bacteriol. 88:99-104. 1964.-Upon release from repression, cells and spheroplasts of two mutants of Escherichia coli efficiently synthesized aspartate transcarbamylase and ornithine transcarbamylase, whereas only cells synthesized dihydroorotic dehydrogenase. Ethylenediaminetetraacetate treatment and sucrose incubation of cells were found to be responsible for the loss of dihydroorotic dehydrogenase synthesis. Spheroplasts required the addition of amino acids and an energy source for the synthesis of aspartate transcarbamylase. Uracil repressed synthesis of aspartate transcarbamylase in spheroplasts as well as in cells. Chloramphenicol inhibition and amino acid requirement for increased aspartate transcarbamylase activity in spheroplasts indicated de novo protein synthesis. E. coli 15, R185-482, and E. coli K-12, 496, were used to study the effect of carbon source and stimulation by orotate and dihydroorotate on synthesis of dihydroorotic dehydrogenase. Only E. coli 15, R185-482, showed any stimulation of dihydroorotic dehydrogenase synthesis. When glucose was the carbon source, orotate but not dihydroorotate stimulated; with glycerol as carbon source, dihydroorotate stimulated and orotate acted as a repressor. These results are discussed in terms of induction and pyrimidine supply to the cells.

Amino Acids↗
PubMed1964

SOME CYTOLOGICAL AND PATHOGENIC PROPERTIES OF SPHEROPLASTS OF CANDIDA ALBICANS.

Kobayashi, George S. (Tulane University, New Orleans, La.), Lorraine Friedman, and Judith F. Kofroth. Some cytological and pathogenic properties of spheroplasts of Candida albicans. J. Bacteriol. 88:795-801. 1964.-Spheroplasts of Candida albicans were prepared by use of an enzymatic mixture from the digestive tract of the snail Helix pomatia. Untreated cells exhibited well-defined cell walls, whereas such structures were absent from spheroplasts. The intravenous inoculation of either spheroplasts or intact cells into rabbits produced a fever which was apparent within 30 min, the "immediate" fever response characteristic of microbial endotoxin. Cell-wall fragments of enzyme-treated cells did not induce a convincing pyrogenic response. When the inoculum was viable, body temperatures did not return to normal but remained elevated until death of the animal 1 or more days later, exhibiting the "delayed" fever of infection. The gross pathological picture in animals succumbing to infection by viable spheroplasts was similar to that obtained with untreated yeast cells.

Animals↗
PubMed1965

FORMATION OF SPHEROPLASTS FROM BACILLUS ANTHRACIS.

Chatterjee, B. R. (Baylor University College of Medicine, Houston, Tex.), and Robert P. Williams. Formation of spheroplasts from Bacillus anthracis. J. Bacteriol. 89:1128-1133. 1965.-Spheroplasts were prepared from Bacillus anthracis by combined treatment with lysozyme and glycine. Glycine, at a final concentration of 3%, was added to cultures of B. anthracis in nutrient broth that had grown at 37 C for 16 to 18 hr under 50% CO(2). After additional incubation under CO(2) for 2 hr, lysozyme, at the appropriate concentration (50 to 100 mug/ml), and sucrose, to a concentration of 15%, were added, and incubation was continued for 2 to 6 hr in CO(2). At the end of this period, incubation in CO(2) was discontinued. Spheroplasts formed after incubation in air for 6 to 12 hr. Lysozyme alone exhibited the same effect when added at much higher concentrations (500 to 2,000 mug/ml) to cultures growing under CO(2). No spheroplasts formed when cultures were treated with glycine alone. Treatment with lysozyme was more effective on smooth strains than rough. Cells from young cultures were more susceptible to lysozyme than older cells. CO(2) apparently was essential for formation of spheroplasts from B. anthracis.

Bacillus anthracis↗
PubMedDate not supplied

[Effect of lysozyme, ethylenediaminetetraacetate, magnesium and mannittol on spheroplast formation in Anacystis nidulans].

Three periods (the lag period and periods of intensive and decelerated spheroplast formation) can be detected in the action of lysozyme on the cells of Anacystis nidulans; this seems to be due to peculiarities in the cell wall structure of the cyanobacterium and heterogeneity of the culture. EDTA at concentrations of 0.175--0.7 mM has effect on the duration of the lag period but increases the rate of spheroplast formation during the subsequent periods. However, the action of EDTA in complex with lysozyme for 3 hours causes lysis of some of the spheroplasts being formed. The concentration of mannitol below 0.4 M decreases the yield of spheroplasts. The level of intact spheroplasts prepared from A. nidulans suggests that at least part of them is capable of reversing into whole cells.

Cyanobacteria↗
PubMed1977

[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.

Ampicillin↗
PubMedDate not supplied

[Spheroplasts of Proteus studied by scanning electron microscopy, freeze etching and ultrathin sections].

Spheroplasts are formed 15 min after the addition of penicillin. Swellings appear at the end or in the middle of the cell. Buds were found on the surface of the spheroplasts. As was found by scanning electron microscopy, the surface of the spheroplasts is folded and, during later stages, wrinkled. "Expulsion" of mesosomes into the periplasmic space was found, as well as separation of vesicles, formed by the outer and cytoplasmic membranes, from the cell surface. The two fracture faces of the outer membrane are uncomplementary; the convex fracture face is mainly smooth but contains occasional dents of the rod-like shape while the concave face does not contain them. The outer (true) surface of the cell wall is smooth. The similarity and differences between the protoplasts, spheroplasts and L forms are discussed . The protoplasts and spheroplasts may divide, grow and multiply under definite conditions of cultivation, like the L forms do, but they do not possess the well developed intracytoplasmic membrane system typical of the L forms.

Cell Membrane↗
PubMed1985

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.

Calcium↗
PubMed1971

Synthesis of protein and nucleic acid by disrupted spheroplasts of Pseudomonas schuylkilliensis.

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.

Anti-Bacterial Agents↗
PubMed1965

Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli.

Mohan, Raam R. (Warner-Lambert Research Institute, Morris Plains, N.J.), Donald P. Kronish, Roland S. Pianotti, Ray L. Epstein, and Benjamin S. Schwartz. Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. J. Bacteriol. 90:1355-1364. 1965.-Spheroplasts of Bacillus cereus strain T and Escherichia coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incubation in 0.05 m tris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 m sodium acetate buffer osmotically stabilized with 1.6 m sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined. B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereus and E. coli indicated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopimelic acid, glutamic acid, and muramic acid.

Amino Acids↗
PubMed1971

Transfection of Escherichia coli and Salmonella typhimurium spheroplasts: host-controlled restriction of infective bacteriophage P22 deoxyribonucleic acid.

Under proper conditions, one infective center was obtained for 3 x 10(8) molecules of P22 phage deoxyribonucleic acid (DNA) when lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli were transfected in the presence of 25 mug of protamine sulfate per ml. A 3- to 50-fold B-specific and K-specific E. coli restriction of the incoming P22 DNA was observed. When P22 DNA-infected E. coli spheroplasts were plated with infertile r(LT) (+)m(LT) (+)Salmonella typhimurium indicator, an additional 70-fold restriction was observed. In the presence of protamine sulfate, penicillin spheroplasts of S. typhimurium SB1330 could be transfected b P22 DNA with efficiencies sometimes approaching those obtained with the E. coli spheroplasts; thus, facilitation of transfection by protamine sulfate is not limited to E. coli or to lysozyme-ethylenediaminetetraacetic acid spheroplasts. The application of these results to studies of transfection among other genuses and to studies of in vitro host-controlled restriction and modification for the two loci in S. typhimurium and the one locus in E. coli is discussed.

Centrifugation, Density Gradient↗
PubMed1994

Direct transfer of plasmid DNA from intact yeast spheroplasts into plant protoplasts.

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, beta-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 microns-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae spheroplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplasts was treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol, 50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detected in the extract of the plant cells after a three-day culture. The GUS activity was higher than that of a reconstitution experiment in which the mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 x 10(8) of S. cerevisiae spheroplasts, was treated with PEG and high pH-high Ca2+ solution. Moreover, the GUS gene expression was resistant to micrococcal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transferred from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.

Arabidopsis↗
PubMed1980

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative Escherichia coli K12 mutant and from Salmonella typhimurium.

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.

Bacterial Proteins↗
PubMed1993

Low temperature protocol for efficient transformation of Mycobacterium smegmatis spheroplasts.

Spheroplasts of Mycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0 x 10(8) to 1.0 x 10(9) spheroplasts to saturating DNA (1 microgram) for 15 min at 5 degrees C resulted in a transfection efficiency of approximately 0.009% . The transfer of the beta-lactamase marker with DNA purified from strain LM15 to spheroplasts of a beta-lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillin-resistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for beta-lactamase Cell-free extracts of penicillin-resistant transformants contained beta-lactamase activity that ranged from 0.046 to 0.134 micromol of benzylpenicillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation of M. smegmatis.

Mycobacteriophages↗
PubMed2004

Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication.

To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB.

Anti-Infective Agents, Local↗
PubMed1999

Variations in mRNA transcript levels of cell wall-associated genes of Saccharomyces cerevisiae following spheroplasting.

mRNA transcript levels of 38 genes from Saccharomyces cerevisiae were investigated during attempted spheroplast regeneration. Many of the genes selected are involved in cell wall biosynthesis. Spheroplasts did not regenerate into osmotically competent cells during the experiment. However, at a mRNA level, the quantities of transcripts were altered between the experimental and control populations. KRE11, EGT2 and MSS10 had their transcript levels increased by more than 10-fold during attempted spheroplast regeneration. A further six genes, FLO1, TIR1, SED1, HKR1, YGR189 and MUC1, showed transcript level increases of at least 5-fold. Five genes showed a change in transcript levels from an undetectable level to a detectable level: SKT5, KRE1, KRE5, SEC53 and DHS1. PMT2 showed a rapid decrease in mRNA levels followed by an increase to the basal level. Thus, cell stress genes, biosynthetic genes and some glycosylphosphatidylinositol-anchored cell wall proteins have their transcript levels increased in regenerating spheroplasts, but their transcription was not sufficient to initiate the replacement of the cell wall in liquid medium.

Cell Wall↗
PubMed1978

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.

Bacterial Chromatophores↗
PubMed1965

LYSIS OF BACTERIAL PROTOPLASTS AND SPHEROPLASTS BY STAPHYLOCOCCAL ALPHA-TOXIN AND STREPTOLYSIN S.

Bernheimer, Alan W. (New York University School of Medicine, New York, N.Y.), and Lois L. Schwartz. Lysis of bacterial protoplasts and spheroplasts by staphylococcal alpha-toxin and streptolysin S. J. Bacteriol. 89:1387-1392. 1965.-Protoplasts of Bacillus megaterium, Sarcina lutea, and Streptococcus pyogenes, and spheroplasts of Escherichia coli were lysed by staphylococcal alpha-toxin, whereas spheroplasts of Vibrio metschnikovii and V. comma were not. In the spectrum of its lytic action, streptolysin S qualitatively resembled staphylococcal alpha-toxin except for failure to lyse S. pyogenes protoplasts. In contrast to the two foregoing agents, streptolysin O did not lyse protoplasts and spheroplasts. The observations are interpreted in relation to similarities and differences in lipid composition of bacterial and mammalian cell membranes.

Animals↗
PubMed1986

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.

Aged↗