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PubMed1965

ACTION OF RIBONUCLEASE PREPARATIONS ON VIABLE YEAST CELLS AND SPHEROPLASTS.

Schlenk, F. (Argonne National Laboratory, Argonne, Ill.), and J. L. Dainko. Action of ribonuclease preparations on viable yeast cells and spheroplasts. J. Bacteriol. 89:428-436. 1965.-Recrystallized pancreatic ribonuclease releases more than half of the ultraviolet-absorbing cellular constituents of Candida utilis and Saccharomyces cerevisiae. The presence of salts and buffer interferes with the action of the enzyme on the cytoplasmic membrane, and intracellular constituents show a similar inhibitory effect. Polymerized ribonuclease and conformational isomers cannot penetrate the pores of the yeast cell wall, but they are effective on the membrane of spheroplasts. All forms of the enzyme interfere strongly with the viability of the cells.

Candida↗
PubMed1984

Isolation of spheroplastic forms of Haemophilus influenzae from sputum in conventionally treated chronic bronchial sepsis using selective medium supplemented with N-acetyl-D-glucosamine: possible reservoir for re-emergence of infection.

The isolation rate of Haemophilus influenzae from patients with persistent production of purulent sputum has been increased by the routine use of a selective medium. Nevertheless, some purulent sputum still fails to yield a pathogen. The selective medium was supplemented with N-acetyl-D-glucosamine to encourage primary isolation of colony forming, spheroplastic H influenzae, which reverted to normal forms on subculture. On the basis of in vitro experiments it is postulated that these spheroplastic forms of H influenzae may be induced by inadequate antimicrobial chemotherapy and may be responsible for re-emergence of symptoms in these patients during or shortly after stopping chemotherapy.

Acetylglucosamine↗
PubMed1990

[Spore-spheroplast transformation of Bacillus megaterium].

A new method for transformation of Bacillus megaterium was developed by modification of Chang and Cohen's method. In our method, spore spheroplasts were used as recipient cells instead of the protoplasts of vegetative cells. Longer incubation (60 min) of spore spheroplasts and plasmid DNA before treatment with polyethylene glycol remarkably increased the efficiency of transformation. The frequency of transformation was about 10(4) per microgram of plasmid DNA. A shot-gun-type cloning of chromosome DNA of B. megaterium ATCC 12872 was available in B. megaterium ATCC 19213 strain by this transformation method.

Bacillus megaterium↗
PubMed1978

Kinetic studies of ampicillin action on Escherichia coli and their spheroplasts.

Kinetic studies of ampicillin action were made on exponentially growing Escherichia coli and on E. coli-spheroplasts using a range of inhibitory and subinhibitory concentrations of ampicillin. For each concentration, the value (ko--ka) representing the difference in generation rates of ampicillin-free culture (ko) and the generation rate of the culture with ampicillin (ka) was calculated and plotted against ampicillin concentration. A straight line relation was obtained with E. coli cells, its intersection with the abscissa, where (ko--ka) = o, give the concentration of ampicillin which exerts no inhibitory action on the cells (0.25 microgram/ml). When ka was plotted against ampicillin concentration, the relation was also linear. Its intersection with the abscissa gives the minimum lethal concentration of ampicillin on the bacterial cells (1.05 microgram/ml). With E. coli-spheroplasts such plots were non-linear which means that a different order of reaction was involved. This difference is probably due to a different mechanism of ampicillin action as revealed by the electroscanning microscopy.

Ampicillin↗
PubMed1985

The immortalization of human lymphocytes by spheroplast fusion.

A method for immortalizing animal cells based on the spheroplast fusion technique of Schaffner is being developed. Human lymphocytes have been fused with E. coli spheroplasts containing the plasmid pTSV3, which represents the entire SV40 genome cloned into the EcoRI restriction enzyme site of the plasmid pAT153. The efficiency of transformation was examined using pTSV3 and derivatives which have deletions in parts of the late gene sequences. Although there was a marked increase in the survival time of the lymphocyte cultures after fusion compared with cells which had been mitogen-stimulated but not fused, the cells did not continue to proliferate indefinitely. Attempts are now being made to extend the survival time by culturing the transformed lymphocytes in the presence of feeder cells.

Cell Division↗
PubMed1988

[Isolation and ultrastructure of Yersinia spheroplasts and protoplasts].

The conditions suitable for the cultivation of Yersinia and the inhibition of their cell-wall synthesis have been selected with the aim of obtaining spheroplasts and protoplasts of these microorganisms. Penicillin, streptomycin and lithium chloride have proved to be not very suitable for this purpose as they induce essential changes in the structure of the cytoplasmic membrane in altered Yersinia forms. The addition of 1% of glycine (for Y. pseudotuberculosis), 1-1.5% of methionine in combination with growth stimulators has made it possible to obtain Yersinia spheroplasts and protoplasts with the intact cytoplasmic membrane, thus permitting the isolation and purification of the cytoplasmic membrane fraction.

Bacteriological Techniques↗
PubMedDate not supplied

[Spheroplast study of Thiobacillus ferrooxidans].

Spheroplasts were prepared, under the action of lysozyme (200 and 400 mcg/ml) from the cells of Thiobacillus ferrooxidans grown on the Waksman medium with elementary sulfur after their treatment with 0.5M NaCl solutions and exact exposure to sucrose (0.5M). Intracellular structures were revealed on the spheroplasts which could not be detected on intact cells.

Microscopy, Electron↗
PubMed1984

Effect of silver on whole cells and spheroplasts of a silver resistant Pseudomonas aeruginosa.

Electron micrographs of a silver resistant strain of Pseudomonas aeruginosa grown in the presence of 50 micrograms/ml Ag+ indicate that the surviving cells are resistant at the level of the cytoplasmic membrane. Spheroplasts of the resistant strain were not lysed by 1 h contact with 8 micrograms/ml Ag+, but were 36% and 22% lysed by 1 h contact with 20 micrograms/ml chlorhexidine and 40 micrograms/ml polysorbate 80, respectively. Spheroplasts of a silver sensitive strain of P. aeruginosa were lysed by 22% on contact with 1 microgram/ml Ag+ for 1 h.

Cell Membrane↗
PubMed1984

[Interaction of Rickettsia akari with the host cell in vitro: multiplication, formation of spheroplast-like forms and its destruction in phagolyosomes].

The electron microscopic study of the interaction of R. akari, strain CK, with the monolayer culture of L-cells was made 4 days after inoculation. Rickettsiae multiplied by transverse binary fission immediately in the cytoplasm of the cells and left the cells by gemmation, surrounded with plasmolemma and a fragment of the host cytoplasm. Alongside with multiplying rickettsiae, spheroplast-like rickettsiae and rickettsiae at the stage of destruction were regularly observed in phagolysosomes. The authors suggest that the normal interaction of rickettsiae with the host cell may be realized by three following routes (1) reproduction, (2) destruction in phagolysosomes and (3) formation of altered (anomalous) forms. The ability of the vegetative forms of rickettsiae and chlamydiae to yield spheroplast-like forms (the initial phase of bacterial L-transformation) indicates that these organisms are similar to bacteria and cannot be themselves regarded as L-forms.

Animals↗
PubMed1982

Induction by mitomycin C of recA protein synthesis in bacteria and spheroplasts.

The effect of mitomycin C on the synthesis of recA protein in Escherichia coli has been analyzed in a variety of conditions, using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). In exponentially growing cultures of E. coli AB 1157, the addition of mitomycin C (5 micrograms/ml) promotes a 15-fold increase of the content of recA protein with respect to the basal level. Kinetic analysis of this induction shows that the maximum is reached 60 to 90 min after the addition of the drug and then the level decreases. In an uvrA mutant treated with mitomycin C the level of recA protein reaches a maximum within an hour and afterwards it does not decrease. Treatment of exponentially growing cells with EDTA and lysozyme induces a 3-fold increase of recA protein content, in comparison to the basal level. When such spheroplasts are added with mitomycin or nalidixic acid, a striking increase of the recA protein content in the spheroplast suspension is observed, which tends to level off an hour and a half after the addition of the drugs. The maximum level of recA protein content is five times the level measured after lysozyme treatment, i.e. 15 times the basal level in exponentially growing cells.

Antibiotics, Antineoplastic↗
PubMed1982

[Inhibiting effect of concanavalin A on certain biosynthetic processes in spheroplasts of the yeast Saccharomyces cerevisiae].

The effect of concanavalin A on biosynthesis of nucleic acids, proteins, structural polysaccharides and glycoproteins of the yeast cell membrane and of enzymes having different localization in the cell as well as on other processes occurring in spheroplasts of the yeasts Saccharomyces cerevisiae IBPhM-350 and CCY 21-4-13 were studied. In both yeast strains lectin strongly inhibited total protein synthesis and produced a weaker inhibiting effect on DNA and RNA synthesis. This was accompanied by a decrease of the activity of the majority of already known enzymes (acid phosphatase, invertase, alpha-glucosidase, polyphosphatase, pyrophosphatase, ATPase) and glucose consumption. In addition concanavalin A inhibited the synthesis of structural components of the yeast cell membrane, i.e. mannane and glucane. The data obtained suggest that lectin (50 microgram/ml or higher) has a toxic effect on yeast spheroplasts (or protoplasts).

Cell Membrane↗
PubMed1976

[The influence of bacterial genes recB, recA, exr and polA on T4 phage DNA transfection of lysozyme spheroplasts of Escherichia coli].

The transfection efficiency of T4 phage DNA of Escherichia coli spheroplasts, carrying mutations in four genes, involved in recombination and reparation (recB, recA, exr, polA), is studied. RecB- and recA- mutations increased the phage yield during transfection, and exr- and polA- mutations decreased it. RecA- mutation is suggested to influence on spheroplasts membrane state, resulting in more efficient replication of transfecting phage DNA.

Coliphages↗
PubMed1976

[Biology of spheroplast- and protoplast-like types of L-forms of Escherichia coli K12 converted with penicillin].

A total of 21 strains of stable L-forms were obtained under the action of penicillin on various Hfr and F- strains of E. coli K12. Three morphological types of the L-forms obtained differed by the character of the cell elements, sensitivity to chemical agents, antibiotics and to T4 and T6 phages. Cell wall was revealed in one type of the L-forms, but the rest L-form types were devoid of the cell walls. Reference of the L-forms which preserved the cell wall to the spheroplastic type, and the L-forms devoid of the cell wall to the protoplastic type is suggested. A possibility of L-forms of spheroplastic type obtained from the Hfr strains to retain the sensitivity to f2 phage and to transmit their chromosomes to bacterial recipients in conjugation was demonstrated. The results obtained are discussed.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Production of spheroplasts from Pseudomonas aeruginosa].

The spheroplasts were prepared from the gram-negative bacterium Pseudomonas aeruginosa IBFM-13 upon incubation with lysozyme, but without EDTA. The following conditions were found to be optimal: 20 percent sucrose; lysozyme, 54 mcg/mg protein of the cells; pH 7.6. The spheroplasts, as well as the cells, have a high rate of malate oxidation, but do not oxidize NADH2, the latter fact suggesting that the cytoplasmic membrane remains intact.

Dose-Response Relationship, Drug↗
PubMed1975

Characterization of a stable spheroplast type L-form of Proteus mirabilis D 52 as cell envelope mutant. I. Isolation, growth characteristics, biochemical activities, and sensitivity to bacteriophages.

A stable spheroplast type L-form could be isolated by transferring 627 single colonies and 195 agar blocks with several colonies of unstable L-forms of Proteus mirabilis D 52 on agar media without supplements of penicillin. The L-form grows well on complex and synthetic agar media, however, it failed to grow in any of the liquid media which have been proved. With one exception (formation of acid from maltose) the L-form shows the same bioche mical activities like the parent rod-shaped bacterium. However, the insensitivity for various phages and the failure of DAP in the envelopes demonstrate that there are profound alterations in the biosynthesis and structure of the murein and of the outer wall layers. The results of these investigations and an ultrastructural analysis (Gumpert and Taubeneck 1975) show that the stable spheroplast type L-form LD 52 B of Proteus mirabilis must be considered as a true cell envelope mutant.

Acids↗
PubMed1976

Characterization of a stable spheroplast type L-form of Proteus mirabilis D 52 as cell envelope mutant. II. Electronmicroscopic investigations.

The ultrastructural analysis of a stable spheroplast type L-form of Proteus mirabilis D 52 revealed characteristic alterations in the organization of the cell envelope including defective changes in the cell envelope structure as for instance the loss of a coherent murein layer, the loss of some components in the outer cell wall layer, the formation of small membraneous vesicles at the tips of loose extensions of the cell wall, a decrease in associations and bindings between wall and membrane, an extension of the periplasmic space, an increase in membrane defects, as well as a disturbed cell division causing unusual modes of multiplication and the formation of various intracellular structures like membrane complexes, characteristic sheet-like membraneous bodies, typical inclusion bodies, and defective phage structures, which all could not be observed in normal rod-shaped cells. The results of these investigations and of those given in a previous paper (Gumpert and Taubeneck 1975) show, that the stable spheroplast type L-form LD 52 B must be considered as a true cell envelope mutant in which the biosynthesis and structure of the cell envelope is altered genetically by one or several mutations whereas the main biochemical activities are the same like those of the parent bacterium. The profound alterations in the cell envelope system, however, lead to some changes in the whole cell organization, which apparently in turn cause disorders even in metabolic and biosynthetic processes not directly involved in the biosynthesis of the cell envelope.

Cell Division↗
PubMed1980

Photophosphorylation capacity of stable spheroplast preparations of anabaena.

Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H(2)O --> NADP, H(2)O --> methyl viologen, and H(2)O --> ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H(2)O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O(2) balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9-aminoacridine fluorescence indicates the formation of a Delta pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH(4) (+). In the presence of NH(4) (+) alone, the membrane potential may act as the driving force for photophosphorylation.Increasing amounts of bovine serum albumin protected phosphorylation from uncoupling by silicomolybdic acid. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive water oxidation by silicomolybdic acid provides evidence that the site for 3-(3,4-dichlorophenyl)-1, 1-dimethylurea action is on the acceptor and not donor side of photosystem II in the procaryote Anabaena. It is concluded that stable spheroplasts retain coupled electron transport approaching in vivo rates.

Journal Article↗
PubMed1978

Resistance of spheroplasts and whole cells of Pseudomonas cepacia to polymyxin B.

Sucrose-lysozyme spheroplasts were prepared from two strains of Pseudomonas cepacia and tested for susceptibility to polymyxin B and benzalkonium chloride. Spheroplasts were more susceptible than whole cells to benzalkonium chloride but not to polymyxin B. Disruption of the outer membrane layer was not by itself sufficient to render P. cepacia susceptible to polymyxin B.

Drug Resistance, Microbial↗