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PubMed1988

Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans.

The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of beta-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and beta-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.

Candida albicans↗

PubMed1978

The isolation and characters of L-forms and reversions of Bacillus licheniformis var. Endoparasiticus (Benedek) associated with the erythrocytes of clinically normal persons.

Thirty-eight strains of the Gram-positive bacterium identified as Bacillus licheniformis var. endoparasiticus (Benedek), referred to as BLE, were isolated in various stages of reversion form the L-forms, from 28 out of 100 samples of whole blood or erythrocytes from normal healthy subjects, after prolonged incubation. Similar results were obtained from 100 samples from hospital patients with conditions not usually associated with blood infection. BLE was isolated from only one of 125 samples of plasma, including those separated from infected erythrocytes. Isolates from cultures incubated for up to 4 months were usually in the form of spheroplasts or diphtheroid bacilli; the fully reverted phase, resembling B. licheniformis, with the capacity to form endospores, was isolated occasionally from cultures aged 1--6 months, and it constituted about half the isolates recovered from cultures aged 6--25 months. BLE was isolated in subculture, and with the usual frequency, in previously unopened, primary cultures. It did not occur in 1200 subcultures of 150 control cultures made with autoclaved or irradiated blood cells; it was not detected in the environment of the laboratory or blood-sampling areas, or on the skin or in the respiratory passages of the operators and other persons associated with the laboratory, where typical, saprophytic B. licheniformis was very rare. It is concluded that this Bacillus species exists as an L-form, associated with the erythrocytes of a large proportion of normal persons, as previously recorded by several observers. Some of the morphological variants associated with the L-cycles have in the past been described as different organisms, for example L-forms of various bacteria or mycoplasmas, and the diphtheroid stage has been thought to belong to the genera Corynebacterium and Listeria. The sporogenous stage, although frequently described, has normally been discounted as a contaminant. These observations do not admit of any conclusion in respect of the claims that such bacteria may have a role in arthritis, cancer or other diseases.

Bacillus↗

PubMed1979

Electron microscopy of the L-cycle in Bacillus licheniformis var. Endoparasiticus (Benedek).

Electronmicrographs of sections show that the L-phases of Bacillus licheniformis var. endoparasiticus (Benedek) simulate the various types of micro-organism described by previous workers as associated with ostensibly noninfective conditions, notably cancer and arthritis; e.g., mycoplasmas, mycobacteria, corynebacteria and actinomycetes. The stages of the L-cycle, from spheroplasts through smaller and larger 'diphtheroid' bacteria to the fully reverted sporogenous bacillus, differ from one another mainly in their degree of reconstitution of the cell envelopes. Occurrence in 'diphtheroids' of nonresistant, cell-wall-deficient spore-like bodies, confirms their relationship with the true sporing bacillus stage. Large, swollen forms appear to be mother cells of minute stages.

Bacillus↗

PubMed1984

The morphological response of Pseudomonas aeruginosa to azthreonam, cefoperazone, ceftazidime and N-formimidoyl thienamycin.

The morphological responses of Pseudomonas aeruginosa to N-formimidoyl thienamycin, azthreonam, ceftazidime and cefoperazone were studied by transmission electron microscopy. They were correlated with the results of viable counts and continuous turbidimetric monitoring. N-formimidoyl thienamycin initially caused the formation of abnormally shaped cells which developed into spheroplasts. The other antibiotics caused filamentation of the bacteria, which subsequently underwent lysis. The degree and mechanism of lysis varied between the antibiotics. Exposure to azthreonam also resulted in the deposition of electron-dense intracellular material. As judged by conventional minimum-inhibitory-concentration tests, all the agents exhibited similar activity against the strains of P. aeruginosa tested, with ceftazidime displaying the highest activity. However, N-formimidoyl thienamycin caused the most extensive morphological damage and resulted in the most rapid fall in viable count.

Anti-Bacterial Agents↗

PubMed1985

Comparative growth of Bacteroides species in various anaerobic culture media.

The growth of five species of Bacteroides in four anaerobic culture media was continuously monitored turbidimetrically. Interspecies differences were observed in the growth of Bacteroides spp. in the various media, but growth in Brain Heart Infusion broth supplemented with yeast extract, haemin and menadione, was consistently better than in Wilkins-Chalgren, Thioglycollate or Schaedler broths. Microscopy of cultures grown overnight in Brain Heart Infusion broth showed that the bacteria exhibited normal morphology but most species grown in the other media displayed filamentation or chain formation. Four of the five species grown in Schaedler broth also exhibited spheroplast formation. This morphological change occurred in the stationary phase of growth, was reduced by inclusion of NaCl in the medium and was abolished in Schaedler broth prepared at double the recommended strength.

Anaerobiosis↗

PubMed1999

Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus.

An amphotericin B (AmB)-resistant mutant was isolated from a wild-type AmB-susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 microg ml-1. The acquired resistance of mycelia was specific for polyene-antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild-type. Chemical analysis of the cell wall revealed that levels of alkali-soluble and -insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild-type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild-type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3-alpha-glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.

Amphotericin B↗

PubMed1985

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci.

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep. lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.

Cell Membrane↗

PubMed1975

Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K 12. Purification and properties of the enzyme.

N-Acetylmuramyl-L-alanine amidase activity was detected in Escherichia coli K 12 by usine N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)-meso-[3H]diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N-acetylmuramyl acid and L-alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K 12. The enzyme was purified to near homogeneity. A molecular weight of 39 000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N-acetylmuramyl-L-alanine amidase activity was carried out by investigating several of its properties.

Alanine↗

PubMed1975

Asymmetrical distribution and artifactual reorientation of lipopolysaccharide in the outer membrane bilayer of Salmonella typhimurium.

Labelling of cell walls or outer membranes from Salmonella typhimurium with ferritin-conjugated antibodies directed against the polysaccharide moiety of the lipopolysaccharide gave the following results: 1. Cell walls or outer membranes from which the mucopeptide had been removed by lysozyme digestion at 0 degrees C carried the label on the outer face of the membrane. 2. When the murein layer was removed by either lysozyme or trypsin at physiological temperature (25-37 degrees C) subsequent labelling showed the lipopolysaccharide to be present on both membrane faces. 3. This reorientation could be achieved by a 1-min treatment of the membranes at 37 degrees C. 4. Glutaraldehyde fixation of the outer membranes did not entirely prevent but somewhat inhibited the temperature-induced reorientation process. 5. The same reorientation phenomenon was observed in lysozyme spheroplasts, which were prepared at 37 degrees C and were subsequently lysed in hypotonic medium at 0 degrees C. These observations are discussed as evidence for a transmembrane movement of lipopolysaccharide, which only takes place in areas where the mucopeptide layer is defective, and only when the temperature is sufficiently high to allow such movement.

Antigen-Antibody Reactions↗

PubMed1976

The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity.

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.

Carbohydrate Epimerases↗

PubMed1976

Photosynthetic activities of membrane preparations of the blue-green alga Phormidium luridum.

Techniques have been developed for the isolation of whole spheroplaste and membrane preparations from the blue-green alga Phormidium luridum which are active in photosystem-II-sensitized and photosystem-I-sensitized electron-transport reactions and in photophosphorylation. Attention is called to the necessity of supplying magnesium and phosphate throughout the preparative and assay procedures for obtaining active membranes. Selective washing of a proper membrane preparation is shown to remove a new protein factor, termed protein factor 1, which restores the ability of the preparation ot catalyze photosystem-I-sensitized reactions.

Calcium↗

PubMed1978

Interaction between penicillin and the DD-carboxypeptidase of the unstable L-form of Proteus mirabilis strain 19.

Binding of penicillin to the DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis results in the rapid formation of a modified enzyme-inhibitor complex which in turn undergoes rapid decay into reactivated enzyme and an antibiotically inactive penicillin degradation product. Major antibiotic metabolites recovered from such interactions were benzylpenicilloic acid and phenoxymethylpenicilloic acid from benzylpenicillin and phenoxymethylpenicillin, respectively, suggesting a second enzymic function of the DD-carboxypeptidase as a penicillinase of low efficiency. Statistical analyses made with the help of a linear regression program show that the enzyme interacts with the substrate UDP-N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)-D-alanyl-D-alanine and either benzympenicillin or carbenicillin in a non-competitive manner.

Carboxypeptidases↗

PubMed1980

Assembly of mitochondria in yeast. Complementation of mitochondrial and cytosolic products in a temporal sequence in vitro.

1. Sequential transfer of derepressing yeast spheroplasts from a medium containing chloramphenicol to one containing cycloheximide or vice versa, shows that the cytosolically and mitochondrially synthesized products are synthesized independent of each other and accumulate in the absence of their counterparts. 2. This has been demonstrated by immunoprecipitation using specific antisera for cytochrome oxidase and ATPase enzymes. 3. The independently accumulated products have been shown to complement each other for the expression of enzyme activity, upon mixing in vitro. 4. By varying the time of treatment with cycloheximide, thereby allowing the mitochondrial protein synthesis to proceed to different extent, a time sequence in the appearance of the mitochondrially synthesized products is demonstrated.

Adenosine Triphosphatases↗

PubMed1997

Expression of the Zymomonas mobilis gfo gene or NADP-containing glucose:fructose oxidoreductase (GFOR) in Escherichia coli. Formation of enzymatically active preGFOR but lack of processing into a stable periplasmic protein.

Glucose:fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal peptide of 52 amino acid residues. Expression of the gfo gene in cells of Escherichia coli K12, under the control of a tac promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins were unstable and rapidly degraded in the periplasm unless an E. coli mutant was used that carried a triple lesion for periplasmic and outer-membrane proteases. When fusion-protein export was inhibited by sodium azide or carboxylcyanide m-chlorophenylhydrazone, the cytoplasmic precursor forms of the respective preGFOR were not degraded. A major protease-resistant GFOR peptide from the OmpA-GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. mobilis, additional protein factors are required for the transport of NADP across the plasma membrane and/or incorporation of NADP into the GFOR apoenzyme.

Bacterial Proteins↗

PubMedDate not supplied

A carbohydrate-degrading enzyme from Candida albicans: correlation between alpha-glucosidase activity and fungal growth.

Attempts to detect inducible carbohydrate-degrading enzymes from C. albicans were performed using carbohydrate-restricted media. Although alpha-amylase (polysaccharide hydrolase) was not induced from media containing starch or dextrin, alpha-glucosidase (disaccharide hydrolase) was induced from maltose-containing medium. alpha-Glucosidase activity was detected from the intact cells in suspension and from the supernatant of both spheroplasted and mechanically broken cells, but not from the media. Enzyme activity in intact cells was pH independent and increased during logarithmic growth phase and in media containing lower concentrations of maltose. The addition of 5 mM 1-deoxynojirimycin, a competitive inhibitor of alpha-glucosidase, suppressed fungal growth by more than 50%. These results suggest that alpha-glucosidase activity is crucial for fungal growth in medium containing maltose as a sole carbon source.

Candida albicans↗

PubMed2002

Isolation and characterization of Saccharomyces cerevisiae mutants with a different degree of resistance to killer toxins K1 and K2.

Killer toxin K1 of Saccharomyces cerevisiae kills sensitive cells of the same species by disturbing the ion gradient across the plasma membrane after binding to the receptor at cell wall beta-1,6-glucan. Killer protein K2 is assumed to act by a similar mechanism. To identify the putative plasma membrane receptors for both toxins we mutagenized three sensitive S. cerevisiae strains and searched for clones with killer-resistant spheroplasts. The well diffusion assay identified three phenotypically different groups of clones: clones resistant simultaneously to both toxins, clones with lowered sensitivity to only K1 toxin and those with strongly lowered sensitivity to K2 and partially lowered sensitivity to K1 toxin. These phenotypes are controlled by recessive mutations that belong to at least four different complementation groups. This indicates certain differences at the level of interaction of K1 and K2 toxin with sensitive cells.

Drug Resistance, Microbial↗

PubMed1997

Ca(2+)-transporting ATPase(s) of the reticulum type in intracellular membranes of Saccharomyces cerevisiae: biochemical identification.

Several lines of evidence are presented to show that the Ca(2+)-ATPase activity of total yeast membranes is due to the reticulum (R) type of Ca(2+)-ATPase: (1) Neither calmodulin nor low concentrations of calmodulin antagonists change Ca2+ uptake; (2) removal of plasma membranes (PM) following Con A treatment of spheroplasts (SP) does not significantly alter Ca2+ uptake by the remaining membranes, but increases its specific activity 3.5-fold; (3) after incubation of membranes with [gamma-32P]ATP, SDS-PAGE shows the formation of acyl phosphate intermediates with molecular masses of around 100, 180-190 and 205 kDa; formation of these acyl phosphates requires Ca2+ and is blocked by cyclopiazonic acid, La3+ ions and in the absence of Ca2+. The data on fractionation of yeast membranes are consistent with the suggestion that both the ER and the Golgi are equipped with Ca(2+)-ATPase(s).

Calcium↗

PubMed1994

Attenuation of fungal virulence by synthetic infectious hypovirus transcripts.

Noninfectious, cytoplasmically transmissible viral double-stranded RNAs of the genus Hypovirus cause reduced virulence (hypovirulence) in the chestnut blight fungus Cryphonectria parasitica, providing the basis for virus-mediated biological control of a fungal disease. Synthetic transcripts corresponding to a full-length hypovirus RNA coding strand are infectious when introduced into fungal spheroplasts by electroporation. Hypovirus infections were readily established in Cryphonectria parasitica and in related fungal species not previously reported to harbor viruses. These results demonstrate the use of a synthetic mycovirus transcript to expand fungal host range, thereby broadening the potential application of virus-mediated hypovirulence to control fungal pathogenesis.

Ascomycota↗