Effect of aculeacin A, a wall-active antibiotic, on synthesis of the yeast cell wall

A wall-active, amphophilic antibiotic aculeacin A significantly but incompletely inhibited in vitro the activity of beta-(1,3)glucan synthase prepared from highly susceptible yeasts Saccharomyces cerevisiae and Candida albicans. In contrast, comparable cell-free preparations from S. cerevisiae active in chitin synthase or mannan synthase were insensitive to the antibiotic, suggesting selectivity of its action in synthesis of the yeast cell wall. An electron microscopic study of the effects of aculeacin A at 0.31 micrograms/ml, the optimally active concentration, on osmotically stabilized C. albicans cells revealed morphological alterations in both cell walls and cell membranes. Deformation in contour and derangement of the layered structure of the cell wall were prominent. In addition, massive fibrous material of beta-glucan-like microfibrils was occasionally extruded from the cell surface. Accompanying this effect on the cytology of the cell wall, ultrastructural and functional impairment of the cell membrane was demonstrated by transmission and freeze-fracture electron microscopic techniques. These data suggest that aculeacin A affects synthesis of the yeast cell wall through not only selective blockage of beta-(1,3)glucan synthase, as a result of a primary interaction with the cell membrane, but also inhibition of the fabrication of beta-glucan or other wall components into well-organized cell walls.     

Incorporation of mannoproteins into the walls of aculeacin A-treated yeast cells

Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33000 mannoprotein into the wall network. This was concomitant with the excretion of the latter molecule into the growth medium. Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin. The protoplast walls did lack this component and contained only minor amounts of the 33000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of the 33000 species were immunodetected in the supernatants from treated and untreated protoplasts. These results point to the existence of specific interactions between the glucan network of the yeast cell surface and some of the wall mannoproteins. On the other hand, the presence of a population of SDS-solubilizable mannoproteins in the wall was independent of glucan levels.Abbreviations SDS sodium dodecyl sulphate - YNB Yeast nitrogen base     

Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.     

Use of RAPD-PCR to isolate a species specific DNA probe for Phytophthora cinnamomi

Abstract Aculeacin A and papulacandin B block cell wall regeneration in Candida albicans protoplasts at an intermediate step in which the protoplasts have not yet synthesized the rigid structure of the cell wall and are therefore still osmotically sensitive. In the presence of the antibiotics, total synthesis of glucan is not significantly lowered with respect to control cells, although most of it appears either in the culture medium or in the regenerating wall as alkali-soluble glucan. Thus, it is proposed that echinocandins (such as aculeacin A) and papulacandins may not inhibit glucan synthesis per se but instead inhibit its incorporation into the supramolecular organization of the cell wall.     

Effect of papulacandin B on the cell wall and growth of Geotrichum lactis

Addition of the antifungal antibiotic papulacandin B to an exponential culture of Geotrichum lactis inhibited incorporation of glucose into the alkali-insoluble and alkali-soluble glucan fractions of the hyphal wall, although the rate of growth was practically unaltered. Synthesis of other cell wall components (i.e. galactomannan and chitin) was not affected. Papulacandin B also induced the proliferation of branches along the hyphae which continued to branch dichotomously resulting in a 'colonial' pattern of growth. Aculeacin A, another antifungal antibiotic that inhibited beta-glucan synthesis also caused morphological alterations similar to those described for papulacandin B. Inhibition of beta-glucan synthesis and the altered growth pattern persisted for several hours after removal of the antibiotic. Recovery of beta-glucan synthesis and restoration of the normal pattern of growth occurred simultaneously. Growth of G. lactis in L-sorbose medium also led to inhibition of beta-glucan synthesis and dichotomous branching.     

Gluconimycin an inhibitor of pyrimidine synthesis inBacillus subtilis

Pyrimidine synthesis inBacillus subtilis PCI-219 cells was the primary site of action of the antibiotic gluconimycin as indicated by its arresting the activity of aspartate carbamoyltransferase as well as accumulation of aspartic acid in cultures fortified with gluconimycin. Microbial growth and viable counts were suppressed by the antibiotic whereas glycolysis and aerobic respiration were insignificantly affected. Gluconimycin failed to induce a lytic effect on cell protoplasts while considerable amounts of substances absorbing at 260 nm were released from mierobial cells treated with gluconimycin.     

Localization of beta-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells.

The distribution of beta-glucan synthases between plasma membranes and intracellular membranes of suspension-cultured Italian-ryegrass (Lolium multiflorum Lam.) endosperm cells was examined. Highly purified plasma membranes prepared from protoplasts were only slightly enriched in beta-glucan synthases assayed at 10 microM- and 1 mM-UDP-glucose. Most beta-glucan synthase was associated with intracellular membranes. These membranes were fractionated on a linear sucrose density gradient and were resolved into different membrane fractions containing beta-glucan synthases. Beta-Glucan synthases assayed at 10 microM-UDP-glucose were found in a fraction banding at a density of 1.11 g . cm-3, but most of the beta-glucan synthase assayed at 1 mM-DDP-glucose was at a density of 1.04 g . cm-3.     

Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast

Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae. In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of beta-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein. Consistent with these results, the toxin was inhibitory to the beta-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S. cerevisiae.     

Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca

Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors. Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan). Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3. However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA. Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L). The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied. The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan. Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis. This pathway seems to act antagonistically towards beta-glucan-mediated signaling. As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.     

外源物质对灵芝多糖和β-葡聚糖发酵的影响

研究了14种外源物质（化合物）对灵芝细胞生长和发酵合成多糖和β-葡聚糖的影响。结果表明,连翘水提物（3g/L）对灵芝细胞生长具有显著促进作用;薏苡仁酯（3g/L）对灵芝胞内多糖和β-葡聚糖的合成均具有促进作用;而桔梗水提物、硝酸铈铵、硝酸镨、茉莉酸甲酯和硝普钠对灵芝细胞生长和产物合成均具有抑制作用。进一步通过Box-Behnken试验设计和响应面法分析,建立了添加薏苡仁酯发酵产β-葡聚糖的二次多项式模型,经分析得到产β-葡聚糖的最优条件为：薏苡仁酯添加量10.5g/L、接种量16%、添加时间第88小时、发酵初始pH 7.00。在此条件下获得β-葡聚糖的产量可达(40.67±8.43)mg/L,与未添加薏苡仁酯的对照组相比,提高了41.86%;多糖产量为(0.99±0.21)g/L,与对照组相比,提高了31.99%。结果提示所得添加薏苡仁酯的优化条件可定向诱导灵芝β-葡聚糖的合成,同时也表明在灵芝液体发酵体系中添加薏苡仁酯发酵产多糖和β-葡聚糖具有一定的实用价值。     

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.     

High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts

We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.     

Development of the system for protoplast generation in Streptomyces aureofaciens strains--chlortetracycline producers

Conditions for preparation and regeneration of protoplasts in a commercial strain of the culture producing chlortetracycline and its derivatives were determined. The protoplasting level depended on the conditions of the mycelium cultivation and composition of a regeneration medium. Under the optimal conditions it amounted up to 10(6) protoplasts/ml. A mutant able to form regenerating protoplasts at a rate of 10(9) protoplasts/ml was isolated. An autoinhibition effect in regenerating protoplasts was observed. As a result ofprotoplast generationing, the morphological variation increased and the protoplast antibiotic activity changed within wide ranges. Variants with higher productivity in comparison to that of the initial strain were isolated. Stability of the inherited property of antibiotic production in the variants is being studied.     

Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.     

Biosynthesis of oleandomycin by cultures of Streptomyces antibioticus obtained after regeneration and fusion of protoplasts

The ability of Streptomyces antibioticus strains to synthesize oleandomycin is studied under the effect of regeneration and fusion of protoplasts. The production of strains-regenerants with an increased (by 30-50%) synthesis of oleandomycin is possible. Regenerants of mutants resistant to the proper antibiotic retain a high level of the oleandomycin synthesis more stably. Variations in the antibiotic-production ability are considered in regenerant populations of various generations.     

Isolation and characterization of plasma membranes from the fungus Podospora anserina

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.     

Increase in eremomycin production by regeneration and UV-irradiation of Amycolatopsis orientalis subsp. eremomycini protoplasts]

Protoplast regeneration of Amycolatopsis orientalis subsp. eremomycini producing eremomycin leads to the change of cultural and morphological properties as well as synthesis of secondary metabolites. Formation of plus-variants with enchanced antibiotic production was promoted by UV-irradiation of protoplasts. These plus-variants can be successfully used for repeating protoplasting--UV-irradiation of protoplasts with further increasing of the strain productivity. Finally activity of the initial A. orientalis culture was increased 7-8 times. Proposed method is recommended for the improvement of actinomycetes strains producing antibiotics especially in the case of cultures with poor sporulation.     

Effects of apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis.

1. The novel aminoglycoside antibiotic apramycin is shown to be a potent inhibitor of protein synthesis in bacteria both in vivo and in vitro. 2. In cell-free systems from Escherichia coli programmed with poly(U), apramycin induces translation errors, as assayed by incorporation of leucine, isoleucine and serine, although this effect occurs only to a limited extent. 3. Apramycin inhibits the translocation step of protein synthesis both in vivo, in protoplasts of Bacillus megaterium, and in vitro, in cell-free systems from E. coli. It is proposed that this is the primary inhibitory effect of the drug.     

The methods of protoplast formation and transformation of Streptomyces strains

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.