[Spheroplasts of Mycobacteria. 1. Spheroplast formation with glycine and lysozyme].
Explore the source record for details and available documents.
Bacteriological Techniques↗
SEARCH · Search PubMed
Results for “Spheroplasts”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Staphylococcal spheroplasts and L colonies. IV. Antimicrobial susceptibility of stable methicillin-induced and lysostaphin-induced spheroplasts.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
[Spheroplasts of mycobacteria. 2. Infection of glycine treated mycobacteria and spheroplasts with the phage and its nucleic acid].
Explore the source record for details and available documents.
DNA, Viral↗
Osmotic properties of spheroplasts from Saccharomyces cerevisiae grown at different temperatures.
Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca(2+), protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg(2+), Ca(2+), K(+), and Na(+) compared with spheroplast membranes from cells grown at 15 C. Mg(2+) and Ca(2+) were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.
Calcium↗
Characterization of spheroplast membranes of Neisseria meningitidis group B.
Spheroplast membranes (spheroplast envelopes) of strain 2091 of group B Neisseria meningitidis were prepared by a procedure that included lysozyme treatment of the cells and osmotic lysis of the resulting spheroplasts. Electron microscopy revealed that the membranes consisted of two unit layers, generally parallel to each other. The membrane preparation migrated as a single component in a 40 to 70% sucrose gradient and consisted of 62% protein, 28% lipid, 9% ribonucleic acid, small amounts of carbohydrate, hexosamine, and deoxyribonucleic acid. When 1 or 10 mug (dry weight) was injected intravenously into rabbits, a mild pyrogenic reaction was elicited. In immunodiffusion tests, immune rabbit serum prepared against spheroplast membranes produced three major precipitin lines, with the homologous antigen solubilized with sodium dodecyl sulfate, and a single line with untreated antigen. The immune serum also reacted with a cell wall antigen, and to a lesser extent with some of the cytoplasmic antigens. Succinate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) oxidase activities were found to be associated with the spheroplast membranes. NADH dehydrogenase also was associated with the membranes but was gradually released and recovered in other fractions. Glutamate-oxaloacetate transaminase, glutamate, glucose-6-phosphate, and isocitrate dehydrogenase activities were not found in the membrane preparation. About one-third of these enzymatic activities were recovered in the supernatant fluid after the sedimentation of the spheroplasts and two-thirds were recovered in the cytoplasmic fraction. N-acetylneuraminic acid (NAN)-condensing enzyme and cytidine monophosphate-NAN synthesizing enzyme also were identified in this organism. These enzymes were not associated with the membranes and were recovered from extracts from whole cells, spheroplasts, or cells exposed to osmotic shock, as well as from spheroplast supernatant and shock fluids. It is concluded that the spheroplast membranes of the strain of meningococci used in these studies are typical of those recovered from gram-negative bacteria.
Animals↗
CYTOPATHOGENIC EFFECT OF BRUCELLA SPHEROPLASTS ON MONOCYTES IN TISSUE CULTURE.
Freeman, Bob A. (The University of Chicago, Chicago, Ill.), and Barry H. Rumack. Cytopathogenic effect of Brucella spheroplasts on monocytes in tissue culture. J. Bacteriol. 88:1310-1315. 1964.-Mononuclear phagocytes from guinea pig peritoneal exudates were shown to ingest both normal Brucella suis and spheroplasts prepared from B. suis by treatment with glycine and with penicillin. Quantitative ingestion studies with P(32)-labeled Brucella showed that rough normal Brucella are ingested at a greater rate than are smooth normal Brucella. Spheroplasts prepared from smooth cells were phagocytized at a greater rate than were the normal smooth cells, and spheroplasts prepared from rough Brucella were phagocytized well, although apparently to a lesser extent than from the normal rough Brucella. The degree of phagocytosis of all spheroplasts appeared to reach a peak and then decrease, indicating a release of ingested bacteria; this release of intracellular bacteria is believed to be due to the cytopathogenic effect exerted by the spheroplasts. Direct microscopic observations showed that infection with living spheroplasts prepared from either smooth or rough Brucella destroyed a major portion of the host cells within 4 hr, but that formalin-killed spheroplasts were no more destructive than were normal Brucella. When host cell destruction was assayed by the release of cellular constituents into the medium, it was apparent that host-cell destruction by spheroplasts reaches a significant level within 0.5 hr after ingestion begins, and is almost complete by 4 hr. The implications of these findings in studies on the nature of intracellular Brucella are discussed.
Bacteriophages↗
Spheroplast induction in clinical isolates of Serratia marcescens in the presence of Ca2+ or Mg2+.
Serratia marcescens was easily induced to form spheroplasts by beta-lactam antibiotics in the presence of Ca2+ or Mg2+ without an osmotic stabilizer such as sucrose. The spheroplasts grew in volume, although they could not divide. They were stable for more than 10 h at 37 degrees C in a medium containing a high concentration of antibiotic, and they had the ability to revert to the original bacillary form. Ca2+ was more effective in spheroplast induction than Mg2+. The effect was proportional to the concentration of cations. In 40% of 180 clinical isolates of S. marcescens, more than 40% of the original bacterial cells were induced to form spheroplasts by ceftizoxime in a medium supplemented with 40 mM Ca2+. A high spheroplast induction rate was observed even in medium with 10 mM Ca2+. Few isolates that were supersusceptible to ceftizoxime (MIC, less than 0.2 microgram/ml) were induced to form spheroplasts at a high rate. No difference in spheroplast induction rate or extent between antibiotic-resistant strains and relatively susceptible strains (MIC, greater than 0.2 microgram/ml) was found. The serotype of S. marcescens had no effect on the spheroplast induction rate. Monocations (Na+ and K+) had little effect on spheroplast induction.
Agglutination Tests↗
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.
Antigens↗
Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles.
The polybasic macromolecules DEAE-dextran (diethylaminoethyl-dextran, molecular weight 500000) and poly-DL-lysine (molecular weight 30000-70000) were absorbed with a high affinity by spheroplasts of Candida utilis and subsequently, induced lysis. The extent of lysis of spheroplasts and of the liberated vacuoles was studied under various conditions using alpha-glucosidase activity and soluble arginine as cytoplasmic and vacuolar markers, respectively. Adsorption of polybases was rapidly completed even at 0 degrees C; however, with small doses, lysis was poor at 0-12 degrees C and extensive at temperatures above 12 degrees C. This permitted the completion of adsorption before initiating lysis. The purified vacuoles were also sensitive to polybases though less so than the spheroplasts; however, after lysis of spheroplasts the liberated vacuoles were well protected against the action of polybases. A treatment with polybases which disrupted more than 99% of the spheroplasts left at least 70% of the vacuoles intact. Potassium chloride in high concentrations and calcium chloride in low concentrations inhibited polybase induced lysis of spheroplasts by preventing or even reversing the polybase adsorption. A polyacidic macromolecule, dextran sulfate, could prevent but not reverse the adsorption of polybase and subsequent lysis. Metabolic inhibitors reduced the susceptibility of spheroplasts to polybase induced lysis. Vacuoles isolated from polybase lysed spheroplasts still contained large pools of soluble amino acids, and their ability to transport arginine specifically is a further indication of their functional integrity.
Arginine↗
[The development and the reversion of spheroplasts of a diaminopimelic acid-auxotrophic mutant of Escherichia coli].
1. The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, DAP-, R+TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells. 2. The release of the R-factor mediated periplasmic TEM-beta-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-beta=lactamase activity. The spheroplasts showed a multiple enhancement of TEM-beta-lactamase activity per mg cell protein compared with rod form cells.
Culture Media↗
Inhibition and activation of mannan synthesis in Saccharomyces cerevisiae spheroplast lysates.
Mannan synthetase activity in spheroplast lysates prepared from Saccharomyces cerevisiae was measured by following the incorporation of [14C]mannose from guanosine 5'-diphosphate-[14C]mannose into material precipitable with cold 0.3 M perchloric acid. When enzyme activity was assayed at high concentrations of spheroplast lysate protein (10 mg/ml) in the presence of 7.5 mM MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the spheroplast lysate at 4 degrees C for 16 to 32 h before assay, by repeated freezing and thawing of the spheroplast lysate, or by the omission of MnCl2 from assay mixtures. The addition of ethylenediaminetetraacetic acid or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for spheroplast lysate as the source of mannan synthetase. The supernatant fluid obtained by centrifuging spheroplast lysate at 100,000 x g, when added to assay mixtures containing either spheroplast lysate preincubated at 4 degrees C or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 mM MnCl2 and was destroyed by heating the supernatant fluid at 60 degrees C for 10 min, or by trypsin treatment at 30 degrees C. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in spheroplast lysates may be modulated by preincubation at low temperature or by varying the available Mn2+ concentration.
Edetic Acid↗
Biochemical and cytological observations during the reversing process from spheroplasts to rod-form cells in Escherichia coli.
Hirokawa, Hideo (National Institute of Health, Tokyo, Japan). Biochemical and cytological observations during the reversing process from spheroplasts to rod-form cells in Escherichia coli. J. Bacteriol. 84:1161-1168. 1962.-Spheroplasts of Escherichia coli B-054 were formed by penicillin, and the reversing process from spheroplasts to rod-form cells was examined. The amount of DNA, RNA, and protein in a single spheroplast increased to four to five times more than that of a rod-form cell during the spheroplast formation. Ultraviolet-absorptive particles, probably nuclei, were localized in several regions within a spheroplast. Morphological changes during the process of spheroplast reversion to rod-form cells were precisely followed. The number of rod-form cells, determined by colony counting according to the time course of reversion, increased very rapidly from the beginning of reversion, without a lag phase. From this evidence, it was concluded that a single spheroplast germinates into several rod-form cells.
Bacterial Proteins↗
Reversion of L forms and spheroplasts of Proteus mirabilis.
Altenbern, Robert A. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.). Reversion of L forms and spheroplasts of Proteus mirabilis. J. Bacteriol. 85:269-272. 1963.-Spheroplasts or 3-B type L forms of Proteus mirabilis formed by growth in the presence of penicillin are able to revert to the bacillary form, as judged by regain of resistance to osmotic shock. Reversion takes place in the absence of exogenous nutrients, although at a lesser rate than that observed in a nitrogen-rich medium. Either chloramphenicol or 6-azauracil can completely inhibit reversion of spheroplasts in a penicillin-free medium. By direct measurement, there was no detectable change in total protein content throughout the period of reversion of a spheroplast suspension. Assay for acid-insoluble diaminopimelic acid (DAP) revealed that a large part of the DAP of the cell wall of normal cells was lost when the cells were converted to spheroplasts by exposure to penicillin. Upon transfer to penicillin-free medium, there was a rapid increase in acid-insoluble DAP in the wall of the reverting spheroplast. When spheroplasts were transferred to penicillin-free medium containing chloramphenicol, the acid-insoluble DAP in the wall of the spheroplast continued to decrease. The significance of these results in regard to sites of activity of the antibiotics involved is discussed.
Cell Wall↗
PRODUCTION AND CHARACTERIZATION OF BRUCELLA SPHEROPLASTS.
Hines, William D. (University of Chicago, Chicago, Ill.), Bob A. Freeman, and Gary R. Pearson. Production and characterization of Brucella spheroplasts. J. Bacteriol. 87:438-445. 1964.-Spheroplasts of Brucella were induced by glycine, penicillin, and a combination of the two. These spheroplasts lacked cell-wall endotoxin but did react with antiserum prepared against cell wall and against whole cells. Phase-contrast photomicrographs and electron micrographs are presented, which show no morphological difference between the protoplasts produced by penicillin and by glycine. The glycine and glycine-penicillin spheroplasts were osmotically sensitive, would not reproduce, and only a small percentage reverted to the bacillary form. Penicillin spheroplasts would not reproduce and were not osmotically sensitive. Penicillin spheroplasts showed significant reversion when induced by low concentrations of penicillin, but the rate of reversion decreased with increasing antibiotic concentration. Penicillin and glycine spheroplasts adsorbed brucellaphage at a reduced rate compared with normal Brucella, but spheroplasts produced by the combined action of penicillin and glycine failed to demonstrate brucellaphage adsorption.
Bacteriophages↗
Round shape enlargement of the yeast spheroplast of Saccharomyces cerevisiae by HM-1 toxin.
The effects of HM-1 killer toxin (HM-1) on yeast spheroplasts of Saccharomyces cerevisiae were examined under osmotically stabilized conditions. Prolonged incubation of spheroplasts in nutrient-rich media resulted in an increase in volume, accompanied by aberrant morphological changes. By contrast, spheroplasts were enlarged, maintaining a round shape, when incubated in HM-1 media. The required 50% effective dose of HM-1 was as low as 2.2 x 10(-8) M, and this effect by HM-1 was specific to yeast sensitive to RM-1. Some parts of the enlarged spheroplasts were stable, but the round shape was deformed as HM-1 was removed from the medium. In both the control and HM-1-treated spheroplasts, the total protein and DNA content were increased by approximately three and four times in response to their incubations, respectively. Cytochemical analysis by 4'6-diamidino-2-phenylindol (DAPI) staining showed multiple nuclei. Consistently, actin patches of cells were evenly distributed in both the control and HM-1-treated spheroplasts. A similar enlargement of spheroplasts was observed with lipophilic antifungal compounds, aculeacin A and papulacandin B, but the effects were distinct from those of HM-1 because the spheroplasts resulted in lysis after a long incubation. The molecular mechanism(s) behind this unique observation remains to be studied, but it is clear that HM-1 is an excellent tool for studying yeast cell biology.
Cell Size↗
Kinetic and morphological observations on Saccharomyces cerevisiae during spheroplast formation.
A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.
Animals↗
Genetic recombination in fused spheroplasts of Providence alcalifaciens.
Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.
Cell Wall↗
Susceptibility of whole cells and spheroplasts of Pseudomonas aeruginosa to actinomycin D.
Cells of Pseudomonas aeruginosa suspended in 0.2 M Mg(2+), 20% sucrose, 0.01 M tris(hydroxymethyl)aminomethane, or water partially release lipopolysaccharide. The release of alkaline phosphatase from the periplasmic space and the ability to form spheroplasts on lysozyme treatment is directly related to the lipopolysaccharide released during treatment with 0.2 M Mg(2+), 20% sucrose, or other agents. The synthesis of ribonucleic acid (RNA) by intact cells, magnesium-lysozyme spheroplasts, or 20% sucrose-lysozyme spheroplasts is not sensitive to actinomycin D, whereas RNA synthesis by intact cells or spheroplasts in the presence of ethylene-diaminetetraacetic acid (EDTA) is sensitive to actinomycin D. EDTA alone has an inhibitory effect on RNA synthesis by whole cell, by magnesium-lysozyme spheroplasts, and by 20% sucrose-lysozyme spheroplasts. The experimental data indicate that, although the cell wall is damaged by 0.2 M Mg(2+) or 20% sucrose treatment in the presence of lysozyme, the treated cells or spheroplasts are still resistant to actinomycin D. These results suggest that the cytoplasmic membrane should be considered as the final and determinative barrier to this antibiotic in this organism.
Alkaline Phosphatase↗