The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform
Sergey B. Zotchev - One of the best experts on this subject based on the ideXlab platform.
-
Biosynthesis of the polyene macrolide antibiotic Nystatin in Streptomyces noursei
Applied microbiology and biotechnology, 2005Co-Authors: Espen Fjærvik, Sergey B. ZotchevAbstract:The polyene macrolide antibiotic Nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on Nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in Nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of Nystatin synthesis became available after the gene cluster governing Nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the Nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring “decoration”. The latter data, supported by analysis of the polyene mixture synthesised by the Nystatin producer, helped elucidate the complete Nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel Nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.
-
Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-DeoxyNystatin to Nystatin
Antimicrobial agents and chemotherapy, 2005Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. ZotchevAbstract:The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in Nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized Nystatin at a reduced level and produced considerable amounts of a putative Nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxyNystatin, a Nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both Nystatin and 10-deoxyNystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of Nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of Nystatin and 10-deoxyNystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of Nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
-
in vivo analysis of the regulatory genes in the Nystatin biosynthetic gene cluster of streptomyces noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
Journal of Bacteriology, 2004Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. ZotchevAbstract:Six putative regulatory genes are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient Nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the Nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of Nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of Nystatin biosynthesis.
Manuel Prieto - One of the best experts on this subject based on the ideXlab platform.
-
Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure.
Biochimica et Biophysica Acta, 2006Co-Authors: Liana C Silva, Alexander A. Fedorov, Ana Coutinho, Manuel PrietoAbstract:The selectivity of the antibiotic Nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, Nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on Nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance Nystatin ability to dissipate a transmembrane K + gradient, showing that the presence of additional double bonds in these sterols–carbon C7 and C22, plus an additional methyl group on C-24, respectively–as compared to cholesterol, is fundamental for Nystatin–sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact Nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by Nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).
-
cholesterol and ergosterol influence Nystatin surface aggregation relation to pore formation
Biophysical Journal, 2004Co-Authors: Ana Coutinho, Liana C Silva, Alexander Fedorov, Manuel PrietoAbstract:Nystatin interaction with liposomes mimicking fungal and mammalian membranes (ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles, respectively) was studied by fluorescence spectroscopy. The activity of this antibiotic was also measured using a pyranine fluorescence detected K+/H+ exchange assay. Nystatin mean fluorescence lifetime varied with the antibiotic concentration and ergosterol content (0-30 mol%) of the lipid vesicles. It sharply increased from 5 to 37 ns upon reaching 100 molecules per liposome, reporting Nystatin oligomerization in the membrane. Concomitantly, spectral alterations typical of excitonic coupling were detected and there was a pronounced increase in the initial rate of pore formation by Nystatin. These findings suggest that Nystatin exerts its antibiotic activity via a two-stage mechanism: at low antibiotic concentrations, surface-adsorbed monomeric antibiotic molecules perturb the lipid packing, changing the permeability properties of the ergosterol-rich liposomes. Upon reaching a critical threshold, Nystatin mode of action switches to the classical model of transmembrane aqueous channel formation. In the presence of cholesterol-containing POPC liposomes, neither Nystatin spectroscopic properties, nor the kinetics of K+ efflux varied with the antibiotic concentration suggesting that in this case the first stage of antibiotic mode of action always prevails or the assemblies formed by Nystatin and cholesterol are very loose.
-
Cooperative Partition Model of Nystatin Interaction with Phospholipid Vesicles
Biophysical journal, 2003Co-Authors: Ana Coutinho, Manuel PrietoAbstract:ABSTRACT Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated Nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl- sn -glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, 〈 A 〉. The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in Nystatin mean fluorescence lifetime from ∼7–10 to 35ns, begins to occur at a critical concentration of 10 Nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric Nystatin is anchored at the phospholipid/water interface and suggest that Nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric Nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6±2 antibiotic molecules.
Håvard Sletta - One of the best experts on this subject based on the ideXlab platform.
-
Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-DeoxyNystatin to Nystatin
Antimicrobial agents and chemotherapy, 2005Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. ZotchevAbstract:The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in Nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized Nystatin at a reduced level and produced considerable amounts of a putative Nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxyNystatin, a Nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both Nystatin and 10-deoxyNystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of Nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of Nystatin and 10-deoxyNystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of Nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
-
in vivo analysis of the regulatory genes in the Nystatin biosynthetic gene cluster of streptomyces noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
Journal of Bacteriology, 2004Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. ZotchevAbstract:Six putative regulatory genes are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient Nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the Nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of Nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of Nystatin biosynthesis.
Olga N. Sekurova - One of the best experts on this subject based on the ideXlab platform.
-
Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-DeoxyNystatin to Nystatin
Antimicrobial agents and chemotherapy, 2005Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. ZotchevAbstract:The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in Nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized Nystatin at a reduced level and produced considerable amounts of a putative Nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxyNystatin, a Nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both Nystatin and 10-deoxyNystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of Nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of Nystatin and 10-deoxyNystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of Nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
-
in vivo analysis of the regulatory genes in the Nystatin biosynthetic gene cluster of streptomyces noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
Journal of Bacteriology, 2004Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. ZotchevAbstract:Six putative regulatory genes are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient Nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the Nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of Nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of Nystatin biosynthesis.
Trond E. Ellingsen - One of the best experts on this subject based on the ideXlab platform.
-
Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-DeoxyNystatin to Nystatin
Antimicrobial agents and chemotherapy, 2005Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. ZotchevAbstract:The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in Nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized Nystatin at a reduced level and produced considerable amounts of a putative Nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxyNystatin, a Nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both Nystatin and 10-deoxyNystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of Nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of Nystatin and 10-deoxyNystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of Nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
-
in vivo analysis of the regulatory genes in the Nystatin biosynthetic gene cluster of streptomyces noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
Journal of Bacteriology, 2004Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. ZotchevAbstract:Six putative regulatory genes are located at the flank of the Nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient Nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the Nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of Nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of Nystatin biosynthesis.
-
Effects of nitrogen sources on cell growth and production of Nystatin by Streptomyces noursei.
The Journal of antibiotics, 2000Co-Authors: Einar Jonsbu, Trond E. Ellingsen, Jens NielsenAbstract:Cell growth and production of Nystatin by Streptomyces noursei (ATCC 11455) were investigated on the three different nitrogen sources, ammonium sulphate, ammonium nitrate and sodium nitrate. S. noursei was able to utilise all of the three tested nitrogen sources for the growth and production of Nystatin. High ammonium concentration had a negative effect on production of Nystatin when phosphate and glucose was in excess. There was an increased production of Nystatin when the cultures became ammonium limited. Cultivation with sodium nitrate as the nitrogen source resulted in a prolonged lag-phase for growth and about 50% lower final Nystatin titres compared with cultures grown on nitrogen sources containing ammonium. Nystatin production was shown to be related to the specific growth rate, its production was increased at decreasing specific growth rates.
