Differential Effects of Monovalent and Divalent Ions upon the Mode of Action of the Polyene Antibiotic Candicidin

The polyene antibiotic candicidin is a potent membrane active agent, the action of which can be inhibited by the presence of certain ions. The destruction of the selective permeability of yeast membranes by candicidin allows small molecules to leak into the environment. Loss of intracellular potassium ions inhibits yeast glycolysis. This inhibition may be reversed by extracellular concentrations of potassium or ammonium ions. Monovalent ions did not prevent antibiotic absorption or protect yeast growth from the action of the antibiotic. Divalent ions did not protect yeast glycolysis from the action of candicidin, but were able to reduce antibiotic-induced membrane damage and allowed yeast growth in the presence of antibiotic. It is suggested that divalent ions may interact with membrane sterols creating steric hindrance to subsequent candicidin absorption.     

Localization of the menaquinone-0 alkylating enzyme in the smooth reticulum of chicken liver microsomes

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 microM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit beta-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi+] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi+] and [psi-] lysates. This misreading was enhanced by addition of the polyamine spermidine.     

Ionophores and intact cells. II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg2+ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213

In beta-lactam producing microorganisms, the first step in the biosynthesis of the beta-lactam ring is the condensation of three amino acid precursors: alpha-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, alpha-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.     

The effects of paromomycin on the fidelity of translation in a yeast cell-free system

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 μM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit β-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi⁺] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi⁺] and [psi^?] lysates. This misreading was enhanced by addition of the polyamine spermidine.     

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg²⁺ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Effect of glycerin on the biosynthesis of heliomycin by Streptomyces olivocinereus

Glycerol as the sole carbon source was added to the medium or biosynthesis of heliomycin by Streptomyces olivocinereus and the effect of its concentration on the culture growth and antibiotic production was studied. The culture growth and the amount of the antibiotic synthesized per 1 unit of the fermentation broth were limited by glycerol added in quantities of 0.05 to 1 per cent. Further increasing of the glycerol concentration had no significant effect on the culture growth and antibiotic biosynthesis. The amount of the antibiotic synthesized per 1 unit of the mycelial mass relatively slightly depended on the glycerol concentration. The rate of glycerol consumption by the young 24-hour culture in batch fermentations markedly exceeded that of glycerol consumption by the 48-hour culture. The younger mycelium significantly increased its rate of glycerol consumption when the initial concentration was increased whereas the rate of glycerol consumption by the more mature mycelium did not depend on the initial concentration of the carbon source (within 0.5-2 per cent). The rate of heliomycin biosynthesis practically slightly depended on the initial concentration of glycerol.     

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.     

Use of an aerodynamic foam quencher in the stab cultivation of mycelial fungi

A scheme for automatic impulse foam breaking during biosynthesis of cephalosporin C in 0.63 m3 pilot plant fermenters was developed and tested. It was shown that the use of an aerodynamic foam breaker did not limit the antibiotic biosynthesis by oxygen and promoted stabilization of the foam level in the apparatus without chemical antifoamers and increased air supply.     

Regulation by aromatic amino acids of the biosynthesis of candicidin by Streptomyces griseus

The biosynthesis by Streptomyces griseus of candicidin, an aromatic polyene macrolide antibiotic, was inhibited by L-tryptophan, L-phenylalanine and, to a lesser degree, by L-tyrosine. A mixture of the three aromatic amino acids inhibited candicidin biosynthesis to a greater extent than did each amino acid separately. L-Tryptophan strongly inhibited the incorporation of the labelled precursors propionate or 4-aminobenzoic acid into candicidin. Incorporation of propionate into candicidin was 50% inhibited by 2.5 mM-tryptophan. Inhibition by tryptophan did not require protein synthesis as the same effect was observed in cells in which protein synthesis was prevented by chloramphenicol. The inhibitory effect of L-tryptophan was partially reversed by exogenous 4-aminobenzoic acid suggesting that this effect is exerted at the level of 4-aminobenzoic acid synthase.     

Effect of the aeration and agitation states on tetracycline biosynthesis in semiproduction-capacity apparatus]

The results of the experiments on determination of the effect of aeration and agitation conditions on biosynthesis of tetracycline in the apparatus of semi-production capacity are discussed. It was shown that the antibiotic production level was not connected with the rate of oxygen solution expressed in the sulphite numbers, i.e. this parameter cannot be used as a scaling-up criterion. Accumulation of the antibiotic in the fermentation broth depended on the volume of the air supplied for aeration. It was determined that the level of CO2 dissolved in the fermentation broth did not reach the values having an inhibitory effect on the biosynthetic process.     

Fungal metabolite sulfamisterin suppresses sphingolipid synthesis through inhibition of serine palmitoyltransferase

Sphingolipids and their metabolites are known to modulate various cellular events including proliferation, differentiation, and apoptosis. Serine palmitoyltransferase (SPT) is the enzyme that catalyzes the first step of the biosynthesis of all sphingolipids. Here, we report that a newly identified antibiotic, sulfamisterin, derived from the fungus Pycnidiella sp., is a specific inhibitor of SPT. The chemical structure of sulfamisterin resembles both that of sphingosine as well as a potent inhibitor of SPT, ISP-1 (myriocin). Sulfamisterin inhibited SPT activity with IC(50) = 3 nM in a cell-free lysate prepared from Chinese hamster ovary (CHO) fibroblasts. Sulfamisterin markedly inhibited the biosynthesis of sphingolipids in living CHO cells and in yeast Saccharomyces cerevisiae as monitored by radioactive precursors. Unlike the cell-free experiments, 10 microM sulfamisterin was required for complete inhibition of sphingolipid biosynthesis in intact cells. We also synthesized a series of structural analogues of sulfamisterin and examined their activities both in cell-free and in living cell systems.     

Influence of the culture medium on the production of iturin A by Bacillus subtilis

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.     

Inhibition of yeast exoglucanases by glucosidase inhibitors

Castanospermine, 1-deoxynojirimycin, and N-methyl-1-deoxynojirimycin, three well-characterized inhibitors of the glucosidases involved in the processing of N-linked oligosaccharides, did not affect the biosynthesis or the secretion of exoglucanases (EC 3.2.1.58) from Saccharomyces cerevisiae and Candida albicans but inhibited the activity itself. Regardless of the substrate used, laminarin or p-nitrophenyl beta-D-glucoside (pNPG), all three inhibitors proved to act in a competitive manner. Castanospermine was the most potent inhibitor, with Ki values ranging from 0.16 to 0.5 microM for three different purified yeast exoglucanases. The inhibition caused by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin was poorer, but still significant. By contrast, the glucosidase inhibitors did not show any action on a partially purified endoglucanase (EC 3.2.1.39) Candida albicans. A purified exoglucanase from Basidiomycete QM 806, which was specific for laminarin, was unaffected by castanospermine but it was still inhibited in an uncompetitive manner by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin. The presence of castanospermine in the culture medium of growing yeasts did not have any effect on yeast growth in spite of the fact that, under the conditions used, the external exoglucanase was fully inhibited. None of the yeast exoglucanases hydrolyzed the glucan synthesized in vitro by membrane preparations derived from either yeast. These results support the concept that yeast exoglucanases are glucosidases that also attack laminarin, rather than glucanases capable of attacking pNPG.     

红霉素生物合成的分子生物学

近年来,国外对大环内酯类抗生素生物合成和基因工程的研究非常迅速,不仅认识了许多抗生素生物合成的过程,而且利用基因工程技术改造抗生素生物合成基因,合成了100多种非天然的“天然”抗生素。抗生素生物合成的分子生物学是抗生素基因工程的基础。本全面介绍了五厌内酯类抗生素的代表－红霉素生物合成分子生物学的历史、现状及发展趋势。     

Adsorption of the antibiotic, nisin, to Streptococcus lactis cells

Nizin is produced by Str. lactis, strain MSU. During biosynthesis it is excreted into the fermentation broth and gradually adsorbed on the organism cells. This was confirmed by experiments with an inactive variant of Str. lactis IIa. The cells of this culture adsorbed nizin from "active" fermentation broth. Adsorption of nizin depended on pH of the medium; at pH 2,3 the cells did not adsorbe the antibiotic and at pH 6.6 the amount of the antibiotic adsorbed by the cells was maximum.     

Effect of aeration on antibiotic production byStreptomyces clavuligerus

Summary During the rapid growth phase ofStreptomyces clavuligerus in a 10 litre fermentor, the level of dissolved oxygen (DO) was found to drop to almost zero for a period of approximately 10 h, delaying the appearance of and lowering the production of the antibiotic cephamycin C. Controlling the DO at either 50% or 100% throughout the fermentation did not significantly alter the specific growth rate of the culture, but did elevate final antibiotic levels two- and three-fold respectively. The improved oxygen availability affected antibiotic production both by increasing the rate of specific cephamycin C bisosynthesis and by maintaining this higher rate throughout the production period. These results demonstrate that controlling dissolved oxygen levels close to saturation during periods of rapid growth markedly improves the efficiency and duration of cephamycin C biosynthesis inS. clavuligerus.     

The role of bitoxibacillin components in the manifestation of its biological activity

New methods were developed and applied to quantitative determination of beta-exotoxin and antibiotic activity of delta-endotoxin with respect to Micrococcus spp. in bitoxibacillin (BTB) and the fermentation broths prepared under industrial conditions. The biosynthesis of beta-exotoxin in the period of its maximum accumulation during the fermentation was estimated. It was shown that the primary biological effect of BTB on insects consisted in the actions of beta-exotoxin and delta-endotoxin. Biological activity of each of the entomocidal components of the entomocidal components of BTB did not practically correlated with the number of viable spores. There was a correlation between the antibiotic activity of crystalline B. thuringiensis subsp. thuringiensis solutions and the insecticidal activity of the entomopathogenic preparations. Determination of beta-exotoxin and antibiotic activity of delta-endotoxin might be used as a complex procedure for testing the quality of BTB. The method for estimating antibiotic activity of the crystal solutions allowed one to assay the biological activity of other preparations based on Bacillus thuringiensis non-synthesizing beta-exotoxin.     

Kinetic aspects of the relation of the growth of the producer and the biosynthesis of heliomycin depending on the carbon source in the medium

Kinetic parameters of Streptomyces olivocinereus 11-98 growth and biosynthesis of heliomycin were studied. It was shown that carbon sources such as glycerol, mannitol and ramnose were the most favourable for the antibiotic biosynthesis. These carbon sources belonged to the group of substances providing high growth rates of the culture. Ranging of the culture growth rates and antibiotic production levels revealed a set of carbon sources providing a converse relationship between the growth rate and antibiotic biosynthesis i.e. L-arabinose, potassium gluconate, raffinose and sucrose. It was suggested that these compounds were catabolic type regulators of heliomycin biosynthesis.     

Urease biosynthesis and isolation in Staphylococcus saprophyticus L-1

Experiments were carried out to investigate the effect of organic components of the medium and cultivation conditions on the multiplication rate and urease biosynthesis by Staphylococcus saprophyticus L-1 cells isolated from natural sources. The yeast enzymic hydrolyzate and corn extract were found to be an adequate substitute for the costly organic components--peptone and yeast extract. The substitutes ensured a high level of urease biosynthesis and biomass accumulation. The biomass accumulation was maximum at pH 6.0-7.0 and the urease activity reached maximum at pH 6.0-6.5. The optimum temperature of cultivation was 37 degrees C. Enhanced aeration and constant pH during microbial cultivation in 250 1 fermenters did not increase the biomass accumulation or urease biosynthesis as compared to flask cultivation. The study of urease isolation from the cell extract showed that the ratio of 3 volumes of ethanol to 1 volume of homogenate was optimum and provided the best precipitation of the enzyme. Preliminary thermal treatment of the cell extract increased the urease activity by 2.5 times. In this situation the activity yield was close to 100%.