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PubMed1993

Spheroplast formation and plasmid isolation from Rhodococcus spp.

The genus Rhodococcus comprises aerobic gram-positive actinomycetes that show considerable morphological and metabolic diversity and are known to be involved in the development of plant diseases and degradation of environmental pollutants. We describe a method for cell lysis and large plasmid DNA isolation from Rhodococcus by creating lysozyme susceptible cells by predigestion with the enzyme mutanolysin. Mutanolysin action resulted in the liberation of reducing sugars and free amino acids from the peptidoglycan layers of the cell wall. A 1-h predigestion with mutanolysin followed by a 0.5-h incubation with lysozyme resulted in spheroplast formation. Complete lysis of cells and efficient isolation of intact large plasmid DNA (108 kb) from wild-type Rhodococcus strains was confirmed.

Chromatography, High Pressure Liquid↗
PubMed1994

Response of spheroplasts and chelator-permeabilized cells of gram-negative bacteria to the action of the bacteriocins pediocin SJ-1 and nisin.

We have attempted to bypass the outer membrane (OM) barrier of Escherichia coli and Salmonella typhimurium with pediocin SJ-1 (as compared with nisin) using chelating agents as OM permeabilizers. EDTA, and to less extent EGTA, enabled nisin, but not pediocin SJ-1, to permeate the cell OM of E. coli, to have access to the cytoplasmic membrane and to cause subsequent permeability changes, indicated by an increase in ANS fluorescence intensity and a shift of its emission maximum. Such spectral changes did not occur when, prior to addition, EDTA was saturated with Ca2+ and Mg2+. ANS fluorescence data indicated that, in spite of the fact that pediocin SJ-1 did traverse the EDTA-permeabilized OM of E. coli, it did not cause perturbation of its cytoplasmic membrane and was, therefore, unable to cause cell death. Spheroplasts prepared from E. coli were lysed when treated with nisin but not with pediocin SJ-1. We suggest that the resistance of Gram-negative bacteria to pediocin SJ-1 is due not only to this material's inability to permeate the OM but also (in contrast to nisin) to its inability to interact with the cytoplasmic membrane.

Animals↗
PubMed1983

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells.

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.

Chloroplasts↗
PubMed1984

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts.

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H(+)/e(-) stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.

Journal Article↗
PubMed1989

The K1 Toxin of Saccharomyces cerevisiae Kills Spheroplasts of Many Yeast Species.

The Saccharomyces cerevisiae K1 toxin killed spheroplasts from the genera Candida, Kluyveromyces, and Schwanniomyces. Cells of these organisms were toxin insensitive. The toxin bound poorly to Kluyveromyces lactis cells. In contrast, Candida albicans bound the toxin to an extent similar to that seen with S. cerevisiae. Thus, wall receptors can define toxin specificity and are necessary but not sufficient for toxin action on intact cells.

Journal Article↗
PubMed1970

Conditions affecting the viability of spheroplasts in urine.

Penicillin-induced spheroplasts from Escherichia coli and Proteus vulgaris, which at neutral pH had little resistance to osmotic lysis, retained viability in media of low osmolality at pH 5.0 to 5.5. In the presence of 1,000 IU of penicillin per ml (pH 5.0) in liquid media, E. coli remained in rod form without conspicuous growth. On hypotonic agar media at pH 5.2 with 1,000 IU of penicillin per ml, the bacteria grew out as L-type colonies. These findings are discussed in relation to penicillin therapy.

Journal Article↗
PubMed1970

Increased Resistance of Pseudomonas aeruginosa to Carbenicillin After Reversion from Spheroplast to Rod Form.

Five of six strains of Pseudomonas aeruginosa had at least fourfold increase in resistance to carbenicillian after they reverted from carbenicillin-induced spheroplasts to rod forms. The most susceptible laboratory strain showed the greatest increase in resistance. There was a marked inoculum effect on the minimal inhibitory concentration of carbenicillin when high inocula (10(7) colony-forming units) or very low inocula (10(2) or 10(3) colony-forming units) were used. Population analysis of this reverted strain revealed increase in resistance of the entire population of cells to carbenicillin. However, the degree of resistance was not homogenous. Only a few cells were highly resistant. Comparison of the effect of carbenicillin on the growth of the parent and reverted strains confirmed the increase in resistance of the reverted strain. The resistance remained stable after 42 passages.

Journal Article↗
PubMed1971

Disruption of bacterial protoplasts and spheroplasts by staphylococcal delta hemolysin.

Fractionation of highly purified staphylococcal delta hemolysin by sucrose density gradient centrifugation, Sephadex G-150 and Bio-Gel A-5m gel filtration, carboxymethyl cellulose chromatography, and isoelectric focusing failed to separate the hemolytic, protoplast-lysing, and spheroplast-lysing activities of the preparation. The activities were reduced to a similar extent when delta hemolysin was treated with any of several phospholipids. The same was true when serum or proteolytic enzymes were used as inactivating agents. Cholesterol had no effect. The activities resisted heating at 80 C for 1 hr and were stable for at least 7 days at 5 C in 0.1 n sodium hydroxide, 0.1 n acetic acid, 6 m guanidine hydrochloride, 8 m urea, and 0.1% sodium lauryl sulfate.

Journal Article↗
PubMed1967

Nutrition and metabolism of marine bacteria. XVI. Formation of protoplasts, spheroplasts, and related forms from a gram-negative marine bacterium.

When cells of a marine pseudomonad were washed and suspended in 0.5 m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 m NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up alpha-aminoisobutyric acid by a Na(+)-dependent process.

Aminoisobutyric Acids↗
PubMed1971

Transfection of Escherichia coli spheroplasts. I. General facilitation of double-stranded deoxyribonucleic acid infectivity by protamine sulfate.

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.

Centrifugation, Density Gradient↗