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PubMedDate not supplied

[Spheroplasts of Torulopsis candida yeasts and their oxidizing activity].

The paper describes the conditions in which the spheroplasts of the yeast Torulopsis candida IBFM-Y-127 with a high respiration rate can be isolated. The preliminary incubation of the cells with SH-reagents has to be carried out in a buffer without an osmotic stabilizer, and the incubation in a medium containing 0.6 M KCL, 0.1 M MgSO4, 0.1 MKH2PO4, pH 5.2. In these conditions the cells are incubated with the enzyme from Helix pomatia during 15 to 20 minutes, and the yield of the spheroplasts is 95 to 100 per cent. The spheroplasts oxidize various substrates (glucose, acetate, ethanol) at the same, or even higher, rate as the intact cells.

Candida↗
PubMed1975

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.

Bacteriophages↗
PubMed1990

[Fusion of Erwinia chrysanthemi spheroplasts in an electric fields].

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.

Bacteriological Techniques↗
PubMed1989

A fast spheroplast formation procedure in some 2,5-diketo-D-gluconate- and 2-keto-L-gulonate- producing bacteria.

Calcium 2-keto-L-gulonate (Ca-2-KLG, a key intermediate in vitamin C synthesis) is produced from calcium 2,5-diketo D-gluconate (Ca-2,5-DKG) by a variety of bacteria. A few bacterial species which efficiently convert glucose to Ca-2,5-DKG have been isolated in our laboratory. Our bacterial collection included species that possess the genes for production of Ca-2-KLG from Ca-2,5-DKG; however, the yield of the former is poor. A procedure for the preparation of spheroplasts in Ca-2,5-DKG- and Ca-2-KLG-producing bacteria was developed for the construction of recombinants (fusants), combining the genes for conversion of glucose to Ca-2-KLG efficiently by protoplast fusion. The standard procedure for spheroplast formation in Gram negative bacteria by the Tris-sucrose-EDTA-lysozyme system did not work in the organisms under investigation. The need for an alternative method was necessary. Our results show that, while the Tris-NaCl-EDTA-lysozyme system (pH 8.3) worked very well with bacterial strains of Gluconobacter oxydans (ATCC9937) and Acetobacter melanogenus (NCIM2259), the Tris-sucrose-EDTA-lysozyme system worked well for Erwinia herbicola (ATCC21998), Pseudomonas chlororaphis (NCIM2041) and Corynebacterium species (ATCC31090). However, none of these systems produced spheroplasts in Brevibacterium ketosoreductum (ATCC21914), for which a separate system is under development.

Bacteria↗
PubMed1977

[Spheroplast bacteriocin from strains of the causative agent of pseudotuberculosis and its identification by the antibacterial activity spectrum and the morphology of the inhibition zones].

Capacity for production of bactericin was found in the spheroplasts of the strains of the rodent pseudotuberculosis microbe. The author named it as pseudotuberculocin. The bacteriocin production was not detected in the initial strains of the pseudotuberculosis microbe. Pseudotuberculocin differed in the antibacterial spectrum and morphology of the growth inhibition zones formed on the plates with the indicator strains from the pesticins produced by the strains of the plaque microbe of the marmot and sandword varieties respectively and the cultures of the plaque microbe from the Transcaucasian Upland, as well as from pesticins of the spheroplasts of these strains. Production of a pesticin differing from pesticins of the rod-like forms of these strains by an ability to inhibit the growth of the homologous indicators was shown in the spheroplasts of the plaque microbe of the marmot and sandword varieties. Sensitivity of the plaque microbe strains of the marmot and sandword varieties to the pesticins of the plaque microbe strains and pseudotuberculocin was noted. With an account of the difference in the activity of the tested pesticins and pseudotuberculocin against the strains of the Frederic collection it is recommended to use this property for identifications of pesticins and pseudotuberculocin.

Animals↗
PubMed1980

[Study of the stimulating action of sturin, its fractions, and the internal protein of T2 phage in transfection of E. coli spheroplasts by Lambda phage DNA].

The stimulating action of sturin and its fractions in the transfection of E. coli spheroplasts with DNA of phage lambda has been shown, and the optimum conditions for the infection of sturin-treated spheroplasts with phage DNa have been established. The biological effect was most pronounced in arginine-enriched low-molecular sturin fraction B. The treatment of spheroplasts with the internal protein of phage T2 did not stimulate transfection. These findings suggest that the specificity of the stimulating action of protamine depends on the peculiarities of its amino acid composition, viz. a high content of arginine which is present in protein molecules in the form of blocks made up of 2-6 amino acid residues.

Arginine↗
PubMed1981

[Membrane potential of Pseudomonas aeruginosa spheroplasts having a cyanide-resistant respiration].

The membrane potential (delta psi) of Ps. aeruginosa spheroplasts was measured by a hydrophobic cation--tetraphenylphosphonium (TPP+) under conditions of simultaneous and separate action of the main respiratory chain and cyanide-resistant oxidase. In all cases, oxidation of endogenous substrates by spheroplasts was followed by generation of a membrane potential of the value of 127--156 mv. An addition of 1 mM cyanide did not practically affect the potential value. The discharge of the potential was attained by further addition of dicyclohexylcarbodiimide (DCCD) or valinomycin. The oxidation of the substrates added by the main respiratory chain was accompanied by an additional increase in the potential by 20--30 mv. In this case, an addition of cyanide decreased the potential by the same value. Oxidation of all substrates, except for TMPD+ ascorbate under conditions of functioning of cyanide-resistant oxidase also increased the potential by 15--50 mv. However, in this case cyanide did not practically affect the increase of the potential. The gain in the potential under TMPD + ascorbate oxidation by cyanide-resistant spheroplasts is prevented by cyanide. A question is discussed as to whether the transfer of reducing equivalents directly via cyanide-resistant oxidase is associated with the generation of a membrane potential or its functioning is not connected with the accumulation of energy in the form available for the cell.

Cyanides↗
PubMedDate not supplied

[Electron microscopic and biochemical study of spheroplasts of the blue-green alga Anabaena variabilis].

Electron microscopy of the spheroplasts of the blue-green alga Anabaena variabilis revealed damages induced by lysozyme. Biochemical analysis confirmed the data of electron microscopy that the spheroplasts had lost partly the cytoplasmic content of the cells. DNA was preserved in the spheroplasts though the nucleoid was not detected by electron microscopy.

Cyanobacteria↗
PubMed1993

Genetic recombination by spheroplast fusion in Escherichia coli K12.

Genetic transfer mediated by spheroplast formation and fusion in Escherichia coli was studied. Recombination did not occur from spheroplasts prepared by the combined glycine and lysozyme-EDTA treatment described by Coetzee et al. (1979). In contrast, when bacteria were exposed to a sub-inhibitory concentration of polymyxin B (0.5 microgram/ml) during the spheroplast generation phase, recombinants arose at frequencies of 1 x 10(-8) to 2.6 x 10(-8). The incidence of genetic transfer was further increased by adding 0.01 M CaCl2 to the polyethylene glycol fusion mixture (from 9 x 10(-7) to 9 x 10(-8). Finally, when the effects of both polymyxin B and calcium chloride were combined recombinants arose at frequencies of 3.4 x 10(-6) to 7.3 x 10(-7). These findings suggest that the detergent-like action of polymyxin B in removing a large part of the outer membrane enhances plasma membrane availability where fusion can take place.

Calcium Chloride↗
PubMedDate not supplied

[Coccoid cells and spheroplasts in cultures of the genus Thermus].

The formation of capable of self-reproduction coccoid cells, and of spheroplasts, was studied in the cultures of Thermus ruber and pigmentless Thermus sp. by light and electron microscopy. The primary origination of cocci might be due to non-uniform or multiple cell division. Spheroplasts can be produced either from coccoid or from rod-like cells as a result of autolysis of the glycopeptide layer of the cell wall. The spheroplasts of the cultures belonging to the Thermus genus are characterized by osmotic stability and a peculiar appearance due to a strongly developed periplasmic space and the eccentric arrangement of the protoplast in it.

Spheroplasts↗
PubMed1978

Inactivation and chemical alteration of mating factor alpha by cells and spheroplasts of yeast.

Mating factor alpha isolated from yeast culture filtrates was radiolabeled by lactoperoxidase-catalyzed iodination, with full retention of biological activity. The (125)I-labeled alpha factor bound at low levels to cells of both mating types (a and alpha) but not to spheroplasts. Despite the low level of binding, large quantities of alpha factor activity were lost by incubation with a cells and a spheroplasts, but not with alpha or a/alpha diploid cells. The amount of activity removed from the culture medium was much larger than the amount of (125)I-labeled alpha factor bound to the cells and was correlated with the appearance of radiolabeled derivatives separable by thin-layer chromatography. Upon removal of the cell wall of alpha and a/alpha cells, the spheroplasts acquired the ability to remove alpha factor activity from culture medium, to generate derivatives of alpha factor, and to respond to alpha factor by a morphological alteration resembling the response of a cells. These findings raise the possibility that the specific enzyme capable of altering alpha factor, possibly a peptidase, is associated with both a and alpha cells but is masked by the alpha cell wall. This suggestion is consistent with the observation that the alpha factor activities of G(1) arrest and cell elongation were blocked by preincubation of a cells with the protease inhibitor Trasylol.

Journal Article↗
PubMed1968

Osmotic fragility and viability of lysostaphin-induced staphylococcal spheroplasts.

When Staphylococcus aureus FDA 209P cells were treated with lysostaphin (1 unit/ml) in hypertonic sodium chloride or sucrose environments, viable, osmotically fragile spheroplasts were produced. Turbidimetric studies indicated that 64% (w/v) sucrose or 20 to 28% (w/v) sodium chloride gives maximal protection against lysis of the lysostaphin-treated cells. The NaCl appeared to give greater protection than the sucrose and proved to be much more suitable for viability and related studies. Viability of both shocked and nonshocked treated cells was determined by S. aureus colony counts on agar plates overlayered with the test dilution of the cells suspended in 4 ml of semisolid agar containing 72% sucrose. The difference in the counts represented the number of revertible spheroplasts. Under these conditions, 30 to 50% of the test cells were recovered as osmotically fragile, but revertible, spheroplasts after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This rewere obtained after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This recovery rate fell off rapidly with prolonged exposure. In view of residual turbidity of 30- and even 60-min exposure preparations, it appeared probable that most of the osmotically fragile cells were eventually converted to protoplasts by the prolonged lysostaphin treatment. Osmotically fragile cells were converted to osmotic stability by fixation with 4% (v/v) Formalin.

Anti-Bacterial Agents↗
PubMed1969

Transformation in quasi spheroplasts of Bacillus subtilis.

RECENTLY DEVELOPED DIFFERENTIAL PLATING MEDIA PERMIT THE DISTINCTION OF FOUR CELL TYPES IN INCOMPLETELY PROTOPLASTED POPULATIONS: intact, osmotically insensitive bacilli; osmotically sensitive rods; spheres with adherent wall residues, called quasi spheroplasts; and protoplasts. Such population mixtures were washed free of lysozyme, and then transforming deoxyribonucleic acid (DNA) was added. Transformation was nil in the protoplasts, very low in the residual osmotically insensitive bacilli, and markedly enhanced in both osmotically sensitive rods and quasi spheroplasts. Transformation in the latter two population fractions was reduced, respectively, by about 60% and about 80% by deoxyribonuclease treatment. DNA adhering to the quasi spheroplasts transforms these cells only if they are permitted to resume wall synthesis; when the same cells are plated on a medium where they shed the residual wall and form L colonies, no transformant L colonies are recovered. It is inferred that far-reaching or complete protoplasting blocks all entry of transforming DNA into the cell interior. This may be owing to eversion of mesosomes. Evidence that intact mesosomes may be required for DNA entry is provided by the finding that the recovery of transformants in the intact cell system is sharply reduced on plating media containing 25% gelatin. On such media, cells expel their mesosomes and 75% of them do not re-form any. Our own data and a survey of published results suggest the generalization that partial depolymerization of the cell wall by lysozyme may enhance competence, whereas its complete removal abolishes it.

Bacillus subtilis↗
PubMed1977

Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.

Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.

Adenosine Triphosphatases↗
PubMed1991

Spheroplast-derived membrane vesicles from Rhodobacter capsulatus cells catalyzing nucleotide transport.

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.

Adenosine Diphosphate↗
PubMed1977

Interaction of DNA with DNA binding proteins. III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts.

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.

Bacterial Proteins↗
PubMed1988

Development of an efficient spheroplast transformation procedure for S. thermophilus: the use of transfection to define a regeneration medium.

The conditions for the efficient polyethylene glycol (PEG)-induced spheroplast transformation of three strains of Streptococcus thermophilus have been established. This required the careful optimization of various experimental parameters, the most important being the choice of the lytic enzyme (lysozyme versus mutanolysin), the extent of cell wall digestion and the conditions for the PEG shock which were found to be strain-specific. The transfection assay we had previously developed for S. thermophilus represented a key step and powerful tool in our transformation studies. It allowed individual and stepwise adjustment of the above mentioned factors, but was also compulsory for the establishment of an effective regeneration medium for the strains we examined. Among various potential osmotic protectors tested, raffinose in combination with CaCl2 and MgCl2 was most efficient and routinely supported regeneration with up to 10% efficiency, after PEG treatment. With the spheroplast transformation procedure described in this paper, shuttle vectors and recombinant plasmids could be introduced into three industrial yogurt starters, with maximal efficiencies of 7.5 x 10(4) transformants/micrograms of liposome encapsulated, covalently closed circular DNA. A striking, yet unexplained, reduction in transformation rates was observed when erythromycin rather than chloramphenicol was used as the selecting agent.

Calcium Chloride↗
PubMedDate not supplied

Evidence for the synthesis of peptidoglycan by spheroplasts of Mycobacterium smegmatis ATCC-14468.

Evidence for the synthesis of insoluble peptidoglycan by spheroplasts of Mycobacterium smegmatis ATCC-14468 was obtained by inhibition of synthesis by vancomycin and cycloserine and no inhibition of synthesis by tetracycline. Radioactivity in the peptidoglycan fraction decreased when treated with lysozyme. The ability of spheroplasts to synthesize macromolecular peptidoglycan was half that of whole cells. A relationship was obtained between the amount of intact cell wall present and peptidoglycan synthesized: as the amount of intact cell wall decreased, the ability to synthesize macromolecular peptidoglycan also decreased.

Cycloserine↗