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At least 19 recordsLinked to original sources

PubMed1975

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.

Coliphages↗

PubMed1977

The photochemical and fluorescence properties of whole cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum.

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured. Photosynthesis (O2 evolution) in whole cells; Hill reaction (O2 evolution) with Fe(CN)63- and NADP as electron acceptors (Photosystem II and photosystem II + Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern. On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90% (10%) of the chlorophyll a, 90% (10%) of the carotenoids and 15% (85%) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments; they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction. The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20--40%) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion. The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component. The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.

Cyanobacteria↗

PubMed1977

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts, spheroplast membrane vesicles and chromatophores.

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.

Bacterial Chromatophores↗

PubMed1975

Transfection of Escherichia coli spheroplasts. V. Activity of recBC nuclease in rec+ and rec minus spheroplasts measured with different forms of bacteriophage DNA.

The in vivo activity of the recBC nuclease was assayed by transfection of isogenic rec+ and rec minus spheroplasts with bacteriophage DNA of various origin and structure. The results indicate that the recBC nuclease can limit transfection at several stages during the production of an infective center; such limitations depend primarily on whether the DNA is in, or assumes, a nuclease-sensitive structure. The first stage of limitation can occur when a nuclease-sensitive transfecting molecule enters the spheroplast. Other potential limitation points occur during replication and maturation of the bacteriophage DNA. The initial stage can be bypassed by using recBC nuclease-resistant molecules such as circular forms. Through analysis of results with other DNA structures, we found that in vivo the effects of the double-strand exonucleolytic activity of the recBC nuclease predominated. The effects of the single-strand nuclease activities seem to be modified from those observed for the purified enzyme in vitro (Karu et al., 1974). Inside the cell, the single-strand exonuclease activity is very weak and the single-strand endonuclease activity is abolished almost completely.

Adenosine Triphosphatases↗

PubMed1975

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↗

PubMed1975

[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↗

PubMed1979

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↗

PubMed1977

[Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis].

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.

Biological Products↗

PubMed1979

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.

Mannitol↗

PubMed1977

[Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine].

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.

Coliphages↗

PubMed1976

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.

Biological Transport, Active↗

PubMed1978

Lack of lysogenic induction in "diaminopimelic acid spheroplasts".

As part of an attempt to develop a semi-in vitro system of lysogenic induction, using spheroplasts of Escherichia coli K-12 lysogenic for prophage lambda, we prepared spheroplasts by depriving E. coli dap of diaminopimelic acid (DAP-spheroplasts). DAP-spheroplasts made from E. coli (lambda cI857) were thermally inducible. However, DAP-spheroplasts of E. coli (lambda) were not inducible by UV light. Thus, it appears that a functional cell wall is required for UV induction of prophage lambda.

Coliphages↗

PubMed1979

Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane.

Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30. The rate of incorporation of [3H]arginine into proteins in spheroplasts was about 30% of that of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein. Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein. No prolipoprotein was observed even with a short pulse-labeling with [3H]arginine. On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed. These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope.

Bacterial Proteins↗

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↗

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↗

PubMed1976

An ultrastructural analysis of protoplast-spheroplast induction in Cryptococcus neoformans.

Protoplasts-spheroplasts of a human isolate of Cryptococcus neoformans were prepared using the gut enzyme of Helix pomatia. The induction process, as studied by transmission electron microscopy, occurred in two stages. Early in the induction process, protoplasts-spheroplasts emerged from whole cells through a break in the cell wall-capsule envelope. Later, a gradual dissolution of the entire cell wall occurred releasing the intact protoplast-spheroplast. A comparison of protoplasts-spheroplasts with normal untreated cells revealed that the degree of cellular vacuolation as well as the resolution of cellular organelles was similar.

Cell Wall↗