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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↗
PubMed1973

Transfection of Escherichia coli spheroplasts. 3. Facilitation of transfection and stabilization of spheroplasts by different basic polymers.

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.

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↗
PubMed1974

Transfection of Escherichia coli spheroplasts. IV. Transfection of rec+ and rec minus spheroplasts by native, denatured, and renatured T5 bacteriophage DNA after repair of single-strand breaks by polynucleotide ligase.

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.

Coliphages↗
PubMed1969

Isolation of spheroplast membranes and stability of spheroplasts of Bacillus stearothermophilus.

Spheroplasts were prepared by lysozyme digestion of the cell wall and ruptured by suspension in 0.15 m NaCl, followed by centrifugation at 30,900 x g for 35 min, and by a final suspension in 0.05 m NaCl for 12 to 16 hr at 5 C. The membrane ghosts were washed four times in tris(hydroxylmethyl)aminomethane (Tris) magnesium buffer and once in distilled water. The intact membranes resembled empty sacs with narrow slits in which the cytoplasm was extruded. A 92% recovery of cell membrane was obtained with all membrane preparations. The spheroplasts do not require a stabilizing medium to keep them from rupturing, and they are stable for 2 to 3 hr when exposed to a temperature of 65 C. The membrane content of the cell increases with age of culture (mid-log, 16.5%; late-log, 17.0%; and stationary, 17.6%) and temperature of growth (55 C, 16.5%; and 65 C, 17.8%), and it is unaffected by composition of the growth medium. The ratio of the protein to lipid content of the membrane increases with the complexity of the medium, age of culture (mid-log, 3.65; late-log, 3.91; and stationary, 4.15), and temperature of growth (55 C, 3.65; and 65 C, 5.22). The ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) content of the membranes was 9.0 to 13.7% and 0.3 to 0.8%, respectively. Reducing sugar (determined as glucose) amounts to 0.9 to 1.0% of the membrane weight and did not significantly vary for the different membrane preparations. Medium composition, age of culture, and temperature of growth have no significant effect on the amount of each amino acid in the membrane. Aspartic acid, glutamic acid, alanine, leucine, and lysine are present in the greatest amount and represent 12.9 to 14.1%, 10.4 to 11.3%, 9.6 to 10.3%, 7.7 to 8.8%, and 7.6 to 8.5% of the membrane peptide, respectively. Prior to the rupture of the spheroplasts, 25.0, 15.7, and 50.0% of the protein, RNA, and DNA, respectively, is lost. In potassium phosphate-magnesium buffer without sucrose, 90% of the protein and RNA and 95% of the DNA is lost from the spheroplasts. In the presence of sucrose, the leakage of RNA and DNA is similar to that observed for spheroplasts suspended in Tris magnesium buffer; however, the leakage of protein is 2.4 times greater.

Amino Acids↗
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↗