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PubMed1977

Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli.

The cytoplasmic and outer membranes of gram-negative bacteria can be isolated from spheroplasts, and separated on sucrose density gradients. Lysis of spheroplasts causes extensive membrane fragmentation and since the characteristics of the fragments obtained by different lysis procedures need not be identical, the influence of the disruption method on membrane composition has been examined. Spheroplasts of Escherichia coli J5 were lysed by osmotic shock, which did not significantly separate the cytoplasmic and outer membranes, but resulted in mixed membrane vesicles. Lysis in the French press and by sonication caused extensive membrane fragmentation and separation. Sonication, however, also caused some fusion between fragments of the outer and the cytoplasmic membranes; this intermembrane fusion increased with sonication time. When the cytoplasmic and outer membranes were well separated and intermembrane fusion was minimal or absent, the cytoplasmic and outer membrane fragments were heterogeneous with respect to density and ovarll phospholipid, protein and lipopolysaccharide composition. In addition, cytoplasmic, but not outer, membrane fragments were also heterogeneous with respect to protein composition. It is concluded, therefore, that membrane fragments obtained from the cytoplasmic and outer membranes are heterogeneous independently of the lysis procedures used to obtain these fragments. Possible reasons for this heterogeneity are discussed.

Bacterial Proteins↗
PubMed1978

The effect of osmotic shock on the accessibility of the murein layer of exponentially growing Escherichia coli to lysozyme.

The restricted access of lysozyme to the murein layer of exponential phase Escherichia coli is enhanced considerably by osmotic shock. When cells suspended in Tris/EDTA/sucrose are diluted 11-fold in water or 10 mM EDTA in the presence of lysozyme, their susceptibility to lysozyme increases by a factor of 50--100, for both Escherichia coli JC411 and W3110, grown to the early exponential phase in unsuppleneted or supplemented minimal media, and in Brain Heart Infusion. Since an 11-fold dilution causes lysis of lysozyme spheroplasts, the effects of a 2-fold dilution have also been investigated. A 2-fold dilution of cell suspended in TrisEDTA/sucrose still increases their susceptibility to lysozyme by a factor of 10--50, but the resulting spheroplasts remain intact. EDTA is necessary to permit lysozyme access to the murein layer during the dilution, which is ineffective in the presence of 5 mM MgCl2. These results are discussed in terms of the formation of lysozyme spheroplasts from young Escherichia coli.

Bacteriolysis↗
PubMed1982

Localization of glycerophosphate acyltransferase in Escherichia coli.

sn-Glycero-3-phosphate acyltransferase (EC 2.3.1.15) the first enzyme involved in phospholipid biosynthesis, is known to be associated with the cytoplasmic membrane of Escherichia coli. The localization of this enzyme in the transverse plane of the membrane was investigated by proteolysis of intact and lysed spheroplasts and by inhibition of glycerol 3-phosphate transport into intact cells in the presence of azide. Glycerophosphate acyltransferase was found to be resistant to proteolysis by trypsin in intact spheroplasts, whereas its enzymatic activity could be destroyed completely by trypsin in lysed spheroplasts. These results are in line with a localization of the acyltransferase at the inner aspect of the cytoplasmic membrane. Sodium azide was shown to have no inhibitory effect on glycerophosphate acyltransferase activity. Lack of incorporation of glycero phosphate into the phospholipids of glycerol phosphate transport-negative cells and inhibition of this incorporation in wild-type and glycerol 3-phosphate transport-constitutive cells by azide support a cytoplasmic-oriented localization of the glycerophosphate acyltransferase.

Acyltransferases↗
PubMed1992

Protein transport in the permeabilized cell of Schizosaccharomyces pombe.

We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae. We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus. Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol. A glycoprotein of S. pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S. pombe system, but not in the S. cerevisiae system. This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S. pombe.

Biological Transport↗
PubMed1993

Site-directed mutagenesis in single cells: transitions produced by DNA carrying a single O6-alkylguanine residue.

Using a single burst assay based on a Poisson Distribution, I have determined the mutant virus frequency in single spheroplasts transfected with phi X174 form I' DNA carrying an O6-methyl-, ethyl-, n-propyl- or n-butylguanine residue at position 2401 of the minus strand. One set of experiments was performed with spheroplasts derived from Escherichia coli AB1157, which has normal DNA-repair systems. Of the cells examined after transfection with DNA carrying a methylguanine moiety, 30% produced mutant virus and 12% contained only mutants; with ethylguanine, 55% of the cells had mutants and 41% produced only mutants; with butylguanine, 6% of the cells had mutants and 3% contained only mutants; with propylguanine no mutants were detected in the 33 cells examined. In similar experiments carried out with spheroplasts defective in excision repair (E. coli AB1157 uvrA6) the percentage of cells producing mutant phage after transfection with DNA carrying an O6-butylguanine residue increased from 6 to 21%, and the percentage of cells producing only mutants increased from 3 to 8%; with DNA carrying an O6-methylguanine moiety, the percentage of cells producing mutants decreased from 30 to 6% and the percentage of cells producing only mutants fell from 12% to 0. In order for an individual uvrA cell to produce exclusively mutant phage from a single O6-alkylguanine residue some form of selection must occur during replication because one strand of the transfecting DNA is wild-type, and excision repair, which could lead to a homoduplex with the transition in both strands, is defective in these cells. This selection must also occur in cells with normal DNA repair. The first event in the selection process is critical; if replication of the alkyl-DNA occurs first and if a mutation is produced, then there is a significant probability that the cell will produce only mutant virus regardless of whether or not repair occurs in subsequent events, but the frequency one observes is influenced strongly by the status of the repair systems in the cell.

Bacteriophage phi X 174↗
PubMed1992

Direct DNA hybridization screening of primary yeast transformants in the construction of targeted yeast artificial chromosome (YAC) libraries.

A simple and inexpensive method for direct, large-scale DNA hybridization screening of primary yeast colonies following introduction of foreign DNA into Saccharomyces cerevisiae by spheroplast transformation is presented. An optimally thin layer of standard regeneration agar containing the transformed spheroplasts is spread on selective plates using carefully controlled temperature conditions; the colonies regenerate initially within the thin layer of hardened regeneration agar, but as they grow, greater than 90% of them break the surface and become accessible to direct lifting and screening methods. The technique avoids the manual or mechanized picking of transformants from within the regeneration top agar, does not require specialized (and often expensive) regeneration matrices such as alginate or low-melting-point agarose, and yields transformation efficiencies and screening results identical to those obtained using the standard spheroplast transformation protocol. We demonstrate the utility of this method for the construction of a targeted human-YAC library from a hamster hybrid cell line that contains chromosome 13 as its only human DNA.

Blotting, Southern↗
PubMed2004

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess.

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.

Culture Media↗
PubMed2004

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells.

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.

Aerobiosis↗
PubMed1975

Isolation of DNA from yeast by chromatography on hydroxyapatite.

A simple procedure is described for isolation of purified non degraded total DNA from yeast cells. The procedure involves conversion of the cells into spheroplasts by enzymatic treatment, lysis of the spheroplasts in 8 M urea - 0.24 M sodium phosphate buffer - 0.01 M EDTA (ethylendiamintetraacetic acid, sodium salt) - 1% SDS (sodium dodecyl sulphate), deproteinization of the lysate with chloroform-phenol and separation of the DNA from proteins, RNA and other contaminants by hydroxyapatite chromatography. The yield is about 90% of the DNA in the starting material (spheroplasts).

Chromatography↗
PubMed1999

Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.

The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently.

Anti-Infective Agents↗
PubMed2003

Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine.

OBJECTIVES: Polyquad (Alcon) (polyquaternium-1, PQ-1) and Aldox (Alcon) (myristamidopropyl dimethylamine, MAPD) are two biocides that are used commercially in a contact lens disinfecting solution, namely Opti-Free Express (Alcon) multi-purpose disinfecting solution. Their potential mechanisms of action were investigated against a range of common ocular pathogens. These were Acanthamoeba castellanii (trophozoites and cysts), Aspergillus fumigatus, Candida albicans, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus. METHODS: Three aspects were investigated: the lethal effects of the biocides on the organisms, the leakage of K+ from treated cells, and the lysis of spheroplasts derived from the cells. RESULTS: PQ-1 was found to have predominantly antibacterial activity, and induced K+ leakage from the bacteria and C. albicans. It also caused lysis of spheroplasts of S. marcescens, but not those of C. albicans. MAPD was active against all of the organisms, but showed higher activity against the fungi and amoeba. It induced K+ leakage from A. fumigatus and C. albicans, and like PQ-1, lysed the spheroplasts of S. marcescens but not C. albicans. CONCLUSIONS: The two biocides have different spectra of antimicrobial activity. PQ-1 has mainly antibacterial activity, whereas MAPD was active against all of the test organisms, particularly the fungi.

Acanthamoeba↗
PubMed1977

Luminescence and respiratory activities of Photobacterium phosphoreum. II. Control by monovalent cations.

Luminescent activity of spheroplasts of the cells of Photobacterium phosphoreum was stimulated by Rb+ and K+ and inhibited by Na+ in the medium. Opposite effects of these ions were observed on the rate of O2 consumption of the spheroplasts through the cytochrome and luciferase electron transfer systems. In vitro activities of NADH-FMN reductase and luciferase were only slightly stimulated by Rb+, K+, and Na+, while Na+ exhibited significant activation of the NADH-oxidizing activity of the cells. The redox level of cytochrome b in the spheroplasts during steady-state respiration in an Na+ medium was more reduced, while that of NAD(P) was more oxidized, than those in an Rb+ or K+ medium. Na+ activates the cytochrome electron transfer system at a point between NADH and cytochrome b, and thus has a stimulative effect on cellular O2 consumption. Rb+ and K+ do not show similar activation, but the cellular NAD(P) was brought to a more reduced level, so that in vivo luminescence is stimulated in an Rb+ or K+ medium.

Cations, Monovalent↗
PubMed1980

Immunological properties of the cell envelope components of Vibrio cholerae.

Several immunobiological properties of cell envelope components of Vibrio cholerae such as mitogenicity, antigenicity, adjuvanticity and toxicity were tested in mice. Killed whole bacteria, spheroplasts, lipopolysaccharide and outer membrane proteins possessed mitogenic activity as determined by [3H]thymidine uptake in spleen cell cultures. All these components predominantly stimulated murine bone-marrow derived (B) lymphocytes. The mitogenicity induced by V. cholerae lipopolysaccharide was similar in magnitude to that observed with Salmonella typhimurium lipopolysaccharide. Vibrio cholerae lipopolysaccharide was mitogenic for gut-associated lymphocytes such as those obtained from Peyer's patches and small intestine. Antibody formation at the cellular level was detected by the haemolytic plaque assay. Plaque-forming cells to V. cholerae lipopolysaccharide were only detected when mice were immunized intraperitoneally with intact cells or with spheroplasts. Among the various cell envelope components, lipopolysaccharide alone possessed adjuvant properties as it increased the number of plaque-forming cells to sheep erythrocytes fourfold in mouse spleens. Also, lipopolysaccharide was the only component found to be toxic for the mouse (LD50 0 . 5 mg). Neither spheroplasts nor outer membrane of V. cholerae showed adjuvanticity or toxicity in mice.

Adjuvants, Immunologic↗
PubMed1983

The intracellular localization of Pseudomonas aeruginosa lectins.

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.

5'-Nucleotidase↗
PubMed1975

Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.

Amino Acids↗
PubMed1977

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.

Alkaline Phosphatase↗
PubMed1986

Astromicin-induced membrane damage in Serratia marcescens.

The morphological changes in Serratia marcescens induced by astromicin were determined by a new technique of electron microscopy, a rapid freezing and substitution fixation technique, and a freeze-fracturing technique. Two structural changes were observed. One was damage to the cytoplasmic membrane, and the other was the accumulation of a large electron-dense mass in the cytoplasm. The damage observed in the cytoplasmic membrane was the disappearance of the unit membrane structure from the thin-sectioned profile of the drug-treated bacteria and the loss of the membrane particles from the fractured surface of the membrane. Damage to the membrane was also suggested by the results of examination of the spheroplasts for stability. The spheroplasts prepared from the drug-treated bacteria were unstable in an osmotically controlled buffer. Most of the spheroplasts were lysed within 3 h, whereas those prepared from control cells were stable for more than 15 h. The electron-dense mass in the cytoplasm was usually seen in the polar region of the cell in close contact with the cell membrane. These structural changes were not specific for astromicin but were also found in gentamicin-treated cells.

Aminoglycosides↗
PubMed1984

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.

Animals↗