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At least 685 records · Page 38Linked to original sources
PubMed1975

Influence of osmotic pressure on transformable and non transformable variants of Neisseria meningitidis. 3. Fragility of the cell membranes.

Penicillin spheroplasts from competent (cp+) and incompetent (cp-) variants of Neisseria meningitidis Strain M1 were equally fragile as determined by the osmotic strength necessary for stabilization. Exponential phase cells adapted to growth under increased osmotic pressure and suspended in acetate or phosphate became osmotically fragile within 2-4 hour when stabilized by sucrose plus MgCl2. Spheroplasts were formed during the succeeding 6-12 hours. Cells that were not adapted to growth at high tonicity but exposed to the stabilizing fluid under conditions leading to plasmolysis, developed osmotic fragility more rapidly, but at the same time lysis of the membranes was activated. Under these conditions, membrane lysis occurred far more rapidly in the cp-variant than in the cp+ variant. Membrane lysis was inhibited by MgCl2 and enhanced by CaCl2 in low concentration. The findings support the notion that osmotic stress in N. meningitidis activates membrane lysis as well as cell wall lysis, and that both activations are far more pronounced in the cp- variant. But the relative protection of the cp+ variant cannot be due to higher tensile strength of the membranes.

Acetates↗
PubMed1976

Osmotically induced changes of cell spaces in Neisseria meningitidis competence variants.

The volume of the whole cell and the fraction of the intact cell bounded by the cytoplasmic membrane (protoplast volume) have been measured by dextran and 14C-sucrose exclusion spaces in Neisseria meningitidis competence variants. Increase in external osmotic pressure causes contraction of the protoplast volume. Increasing osmolality due to NaCl and MgCl2 also causes contraction of the volume of the whole cell, whereas increasing concentrations of sucrose cause little or no change in the whole cell volume. The experiments demonstrate a significant difference between competent (cp+) and incompetent (cp-) cells. The cp+ protoplast have a far higher capacity for swelling during decreasing osmolality, and for shrinkage during increasing osmolality. Comparison of spheroplasts obtained by autolysis as well as by the penicillin technique indicates that the average cp+ spheroplast is larger than the average cp- one. The significance of the difference in structure of cp+ and cp- protoplasts has been discussed.

Cell Wall↗
PubMed1979

Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics.

A biochemical method was developed to quantitatively compare the effectiveness of beta-lactams in triggering murein degradation (autolysin activity) in Escherichia coli. Bacteria prelabeled in their cell walls with radioactive diaminopimelic acid in growth medium were exposed for 10 min to the antibiotics at the appropriate minimal growth inhibitory concentrations and at multiples of these values, and the rate of cell wall degradation was followed during subsequent penicillin-binding protein (PBP)-1 were the most effective triggers of autolytic wall degradation; beta-lactams selective for PBP-2 were the poorest; and antibiotics preferentially binding to PBP-3 showed intermediate activities. The relative effectiveness of beta-lactams in autolysin triggering was found to parallel the effectiveness of the same drugs in causing rapid loss of viability, culture lysis, and spheroplast formation. Autolysin triggering was suppressed by inhibitors of protein and ribonucleic acid biosynthesis but not by inhibitors of deoxyribonucleic acid synthesis. The beta-lactam-induced cell wall degradation did not seem to involve a direct stimulation of enzyme activity or synthesis of new enzyme molecules, and murein sacculi isolated from cells that had been preexposed to a triggering dose of beta-lactam treatment exhibited the same sensitivity to crude, homologous autolysins as sacculi prepared from untreated control bacteria. On the basis of these observations, mechanisms are considered for the triggering of E. coli autolysins and for the role of autolytic activity in bacterial spheroplast formation, lysis, and death.

Anti-Bacterial Agents↗
PubMed1990

Involvement of a cell wall receptor in the mode of action of an anti-Candida toxin of Pichia anomala.

Hanes-Woolf, Dixon, and Hill plots of growth rates of Candida albicans RC1 grown in various concentrations of glucose and a Pichia anomala WC65 toxin suggested the presence of toxin-binding sites. Indirect immunofluorescence microscopy with antitoxin antibodies demonstrated binding of the toxin to the cell wall. Resistance to the toxin of a mutant Saccharomyces cerevisiae deficient in cell wall beta-1-6-D-glucan suggests that the glucan either served as the receptor or influenced the number or composition of the receptor. Immunofluorescence that appeared to be associated with the cell membrane of toxin-treated spheroplasts of C. albicans was also observed. Spheroplasts of the resistant mutant of S. cerevisiae were sensitive to the toxin.

Candida albicans↗
PubMed1999

Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization.

Human saliva contains histidine-rich proteins, histatins, which have antifungal activity in vitro. The mechanism by which histatins are able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the spheroplasts was saturable and could be competed with unlabeled histatin 3. A single histatin 3 binding site with a K(d) = 5.1 microM was detected. Histatin 3 binding resulted in potassium and magnesium efflux, predominantly within the first 30 min of incubation. Studies with fluorescent histatin 3 demonstrate that the protein is internalized by C. albicans and that translocation of histatin inside the cell is closely associated with cell death. Histatin binding, internalization, and cell death are accelerated in low-ionic-strength conditions. Indeed, a low extracellular salt concentration was essential for cell death to occur, even when histatin 3 was already bound to the cell. The interaction of histatin 3 with C. albicans, and subsequent cell death, is inhibited at low temperature. These results demonstrate that the candidacidal activity of histatin 3 is not due exclusively to binding at the cell surface but also involves subsequent interactions with the cell.

Binding Sites↗
PubMed1976

Bacteriological study on effects of beta-lactam group antibiotics in high concentrations.

The growth and viability of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa exposed to various concentrations of a number of beta-lactam group antibiotics were determined. In S. aureus, the bacteriolytic and bactericidal activity of these drugs was lower at very high drug concentrations than that occurring at low concentrations, but these phenomena were not observed in E. coli and P. aeruginosa. Under phase-contrast and scanning electron microscopy, S. aureus treated with high concentrations of beta-lactam group antibiotics revealed a lower frequency of bacteriolysis than at low drug concentrations, and similarly by transmission electron microscopy fewer cells were transformed into spheroplasts at high drug concentrations. However, swelling of the cell wall septum was seen in many cells. Spheroplast formation occurred with the highest frequency at drug levels near the minimum inhibitory concentration and became less frequent as drug concentrations were increased.

Anti-Bacterial Agents↗
PubMed1996

Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock.

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope.

Antibodies, Monoclonal↗
PubMed1973

Subcellular localization of the leucine biosynthetic enzymes in yeast.

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.

Acyltransferases↗
PubMed1973

Freeze-etching of the cell envelope of an Acinetobacter species which carries a regular array of surface subunits.

Freeze-etching was applied to preparations, with and without glycerol, of Acinetobacter sp. strain MJT/F5/199A, consisting of intact cells after normal growth or after incubation with chloramphenicol, spheroplasts, and isolated cell walls and outer membranes. Etched preparations show that a regular array of subunits forms the surface of normal cells. Near the zones of constriction in dividing cells, blebs and irregularities are seen, and some blebs, consisting of both surface subunits and outer membrane, are released from the cells. The cross-fractured cell envelope shows four layers which are related to the structures seen in section as follows: cw1, which is not visible in section, contains the surface subunits; cw2 consists of all or part of the outer membrane; cw3 includes the intermediate and dense, peptidoglycan-containing layers; within these cell wall layers is the plasma membrane. Internal fracture of the plasma membrane occurs under all conditions tested, but the fracture plane in the cell wall is only revealed in chloramphenicol-treated cells or normal cells freeze-fractured with glycerol present; the characteristic fracture faces are not seen in spheroplasts or isolated outer membranes. The concave fracture face cw2 consists of densely packed granules, while the convex face cw3 is fibrillar. The probable location of this fracture plane is discussed. After incubation with chloramphenicol, the outer surface of the cells is obscured by extracellular material, the dense peptidoglycan-containing layer is increased in thickness, and the cytoplasm contains rounded bodies bounded by one or more unit membranes.

Alcaligenes↗
PubMed1974

Isolation and characterization of the outer membrane of Neisseria gonorrhoeae.

The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae.

Bacterial Proteins↗
PubMed1974

Action of lysozyme on penicillin-induced filaments of Proteus vulgaris.

Penicillin-induced filaments of Proteus vulgaris P 18 had a seemingly unaltered cell envelope. Lysozyme, however, reduced the wall part of these filaments into a three-layered structure and caused transformation into spheroplasts. Although cross walls and membrane septa were absent in the filaments, most of the lysozyme-induced spheroplasts remained attached to each other.

Animals↗
PubMed1975

Nitrate reductase system in Staphylococcus aureus wild type and mutants.

Respiratory nitrate reductase with lactate as a hydrogen donor has been studied in cells and spheroplast preparations of wild type and heme-deficienct mutants of Staphylococcus aureus. The activity is rapidly induced when suspensions of aerobically grown cells are incubated without aeration in a complete medium with nitrate. In ruptured spheroplast preparations, the activity with lactate as the donor is located in the membrane fraction, whereas at least 50% of the activity assayed with reduced benzyl viologen is in the cytoplasm. The reductase is inhibited by azide and cyanide, and the lactate-linked system is also sensitive to oxamate, 2-heptyl-4-hydroxyquinoline-N-oxide, dicoumarol, and p-chloromercuribenzoate. An inactive form of the reductase is apparently made during induction with tungstate; this can be activated by subsequent incubation with molybdate in the presence of chloramphenicol. Nitrate reductase activity with reduced benzyl viologen as the donor is induced in suspensions of heme-deficient mutants in the presence or absence of heme. The proportion of cytoplasmic activity is increased in the absence of heme. The staphylococcal nitrate reductase has many of the characteristics commonly associated with the respiratory enzyme in other organisms, but the apparent predominance of cytoplasmic activity is unusual.

Benzyl Viologen↗
PubMed1975

Role of the cell surface in bacterial mating: requirement for intact mucopeptide in donors for the expression of surface exclusion in R+ strains of Escherichia coli.

Two derivatives of the F-like R factor R1drd19 carrying mutually exclusive resistance determinants were used to study the role of the mucopeptide in the expression of conjugal functions. The use of metabolically active penicillin spheroplasts in R+ times R- matings had no effect on the ability of the cells to donate or accept a plasmid. However, in R+ times R+ matings it was found that surface exclusion was totally abolished if the donor, but not the recipient, was a spheroplast. This result implies that the traS gene, expressed by the excluding plasmid, is dependent for its action on an intact mucopeptide layer in the donor cell, and that this interaction is independent of the transfer ability of the excluding plasmid.

Bacterial Proteins↗
PubMed1977

Two systems for the uptake of phosphate in Escherichia coli.

Mutants of Escherichia coli K-12 were constructed such that each possessed one single major system for phosphate transport. A comparison of these strains showed that one of the systems (PIT) was fully constitutive, required no binding protein, and operated in spheroplasts. It permitted the complete exchange of intracellular phosphate with extracellular phosphate (or arsenate) and was completely inhibited by uncouplers. The other system, PST, was repressible by phosphate concentrations above 1 mM, required the phosphate-binding protein for full activity, and did not operate in spheroplasts. It catalyzed very little exchange between internal and external phosphate and was resistant to uncouplers. The maximal velocities attained by the two systems were approximately the same, but the affinity for phosphate in the PST system was greater by two orders of magnitude. In strains in which both systems were fully operative, the initial rates of uptake was nearly additive, and the systems appeared to interact with a common intracellular phosphate pool.

Arsenates↗
PubMed1978

Periplasmic localization of nicotinate phosphoribosyltransferase in Escherichia coli.

Nicotinate phosphoribosyltransferase (NAPRTase) in Escherichia coli mediates the formation of nicotinate mononucleotide, a direct precursor of nicotinamide adenine dinucleotide (NAD), from nicotinate and 5-phosphoribosyl-1-pyrophosphate. Specifically, NAPRTase contributes to NAD synthesis by utilizing intracellular nicotinate formed from NAD degradation products, which are recycled by NAD cycle enzymes and exogenous nicotinate when it is available. In previous studies, it has been tacitly assumed that almost all NAD cycle enzymes are localized in the cytoplasm of E. coli. The results of this investigation provide evidence that NAPRTase is a periplasmic (extracytoplasmic) enzyme. The osmotic shock of exponential-phase cells of E. coli K-12 and ML 308-225 resulted in the release of 63 to 72% and 42 to 48%, respectively, of the NAPRTase into the shock medium. In addition, when exponential cells of strains K-12 and ML 308-225 were converted into spheroplasts, 75 to 84% and 54 to 68%, respectively, of the enzyme was released into the spheroplast medium. Since previous estimates of the effective levels of NAPRTase present in putative repressed and derepressed E. coli cells appeared to be very low, a more convenient and accurate alternative method for the evaluation of NAPRTase in whole cells was developed. The results show that NAPRTase is subject only to a modest degree of enzyme repression. In addition, no evidence was found for the presence of a protein or low-molecular-weight inhibitor of the enzyme in repressed cells.

Cytoplasm↗
PubMed1980

High-frequency heterokaryon formation by Mucor racemosus.

Heterokaryons from Mucor racemosus were produced from two auxotrophic strains of the fungus. Germlings were converted to spheroplasts by using commercial chitinase and purified Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed and fused in solutions of polyethylene glycol and CaCl2. Under optimal conditions, prototrophic heterokaryons were formed at a frequency of about 5%.

Calcium Chloride↗