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Ippolito Natale Camele - One of the best experts on this subject based on the ideXlab platform.
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An attempt of biocontrol the tomato-wilt disease caused by Verticillium dahliae using Burkholderia gladioli pv.agaricicola and its bioactive secondary metabolites
International Journal of Plant Biology, 2017Co-Authors: H S Elshafie, Sabino Aurelio Bufo, Shimaa H. Sakr, Ippolito Natale CameleAbstract:There is a great interest in discovering new microbial natural biocides such as microbial secondary metabolites to reduce the environmental pollution due to the excessive use of synthetic pesticides. Verticillium wilt, caused by the soil-borne Verticillium dahliae, is a widespread disease in tomato growing in many parts of the world. Burkholderia gladioli pv. agaricicola produces some antimicrobial substances and extracellular hydrolytic enzymes which exhibited promising antimicrobial activity towards several phytopathogens. The aims of the current research are to assess in vitro fungicidal effect of 4 strains of B. gladioli pv. agaricicola (ICMP11096, 11097, 12220 and 12322) against V. dahliae using culture or cell-free culture filtrate. In situ assay was performed to evaluate the biocontrol effect of the most efficient bacterial strain on wilt disease caused by V. dahliae in tomato plants. Results demonstrated that the studied bacterial strain ICMP12322 exerted the highest in vitro antifungal activity against V. dahliae which correlated with its ability to produce extracellular hydrolytic enzymes. Furthermore, in situ results showed that the selected bacterial strain significantly minimized the disease incidence.
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In vitro study of biological activity of four strains of Burkholderia gladioli pv. agaricicola and identification of their bioactive metabolites using GC-MS.
Saudi Journal of Biological Sciences, 2017Co-Authors: H S Elshafie, R Racioppi, Sabino Aurelio Bufo, Ippolito Natale CameleAbstract:This research was carried out to study in vitro antibacterial activity of 4 strains of Burkholderia gladioli pv. agaricicola (Bga) against G+ve Bacillus megaterium and G−ve Escherichia coli, haemolytic activity against the cell membrane of erythrocytes, the production of extracellular hydrolytic enzymes and finally, the pathogenicity against Agaricus bisporus flesh blocks. Chemical structure of bioactive substances of the most bioactive strain (ICMP 11096) was established using gas chromatography–mass spectrometry (GC–MS). All the studied Bga strains inhibited the growth of the two tested bacteria although some growing substrates negatively influenced the antimicrobial substance production. The same Bga strains showed highly haemolytic activity and were able to produce 3 hydrolytic enzymes, i.e. chitinase, glucanase and protease. In pathogenicity assays, the considered Bga strains resulted virulent for A. bisporus. The GC–MS for compounds from Bga ICMP 11096 were compatible with the structure of two bioactive fatty acids identified as methyl stearate and ethanol 2-butoxy phosphate with mass spectrum m/e 298 and 398, respectively.
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In Vitro Antifungal Activity of Burkholderia gladioli pv. agaricicola against Some Phytopathogenic Fungi
International Journal of Molecular Sciences, 2012Co-Authors: H S Elshafie, Nicola Sante Iacobellis, R Racioppi, Ippolito Natale Camele, Laura Scrano, Sabino Aurelio BufoAbstract:The trend to search novel microbial natural biocides has recently been increasing in order to avoid the environmental pollution from use of synthetic pesticides. Among these novel natural biocides are the bioactive secondary metabolites of Burkholderia gladioli pv. agaricicola (Bga). The aim of this study is to determine antifungal activity of Bga strains against some phytopathogenic fungi. The fungicidal tests were carried out using cultures and cell-free culture filtrates against Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. Results demonstrated that all tested strains exert antifungal activity against all studied fungi by producing diffusible metabolites which are correlated with their ability to produce extracellular hydrolytic enzymes. All strains significantly reduced the growth of studied fungi and the bacterial cells were more bioactive than bacterial filtrates. All tested Bulkholderia strains produced volatile organic compounds (VOCs), which inhibited the fungal growth and reduced the growth rate of Fusarium oxysporum and Rhizoctonia solani. GC/MS analysis of VOCs emitted by strain Bga 11096 indicated the presence of a compound that was identified as 1-methyl-4-(1-methylethenyl)-cyclohexene, a liquid hydrocarbon classified as cyclic terpene. This compound could be responsible for the antifungal activity, which is also in agreement with the work of other authors.
Gregory L. Challis - One of the best experts on this subject based on the ideXlab platform.
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Genomics-driven discovery of a novel glutarimide antibiotic from Burkholderia gladioli reveals an unusual polyketide synthase chain release mechanism.
Angewandte Chemie International Edition, 2020Co-Authors: Gregory L. Challis, Matthew Jenner, Joleen Masschelein, Isolda Romero-canelón, Yousef Dashti, Ioanna T. Nakou, Eshwar MahenthiralingamAbstract:A gene cluster encoding a cryptic trans -acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters enabled gladiostatin, the metabolic product of the PKS, to be identified. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, indicating it plays a key role in polyketide chain release and butenolide formation.
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genomics driven discovery of a novel glutarimide antibiotic from Burkholderia gladioli reveals an unusual polyketide synthase chain release mechanism
Angewandte Chemie, 2020Co-Authors: Ioanna T. Nakou, Matthew Jenner, Joleen Masschelein, Yousef Dashti, Eshwar Mahenthiralingam, Gregory L. Challis, Isolda RomerocanelonAbstract:A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.
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Discovery and biosynthesis of gladiochelins: unusual lipodepsipeptide siderophores from Burkholderia gladioli
2020Co-Authors: Yousef Dashti, Ioanna T. Nakou, Alex J. Mullins, Gordon Webster, Xinyun Jian, Eshwar Mahenthiralingam, Gregory L. ChallisAbstract:Abstract Burkholderia is a genus of diverse Gram-negative bacteria that includes several opportunistic pathogens. Siderophores, which transport iron from the environment into microbial cells, are important virulence factors in most pathogenic Burkholderia species. However, it is widely believed that Burkholderia gladioli, which can infect the lungs of cystic fibrosis (CF) sufferers, does not produce siderophores. B. gladioli BCC0238, isolated from the lung of a CF patient, produces two novel metabolites in a minimal medium containing glycerol and ribose as carbon sources. HPLC purification, followed by detailed spectroscopic analyses, identified these metabolites as unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The absolute configurations of the amino acid residues in the two metabolites was elucidated using Marfey’s method and the gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of most B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S (CAS) assay showed the metabolites bind ferric iron and that this supresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes. Together, these observations suggest that these metabolites likely function as siderophores in B. gladioli.
Camele, Ippolito Natale - One of the best experts on this subject based on the ideXlab platform.
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In vitro study of biological activity of four strains of Burkholderia gladioli pv. agaricicola and identification of their bioactive metabolites using GC–MS
'Elsevier BV', 2017Co-Authors: Elshafie, Hazem S., Racioppi Rocco, Bufo, Sabino Aurelio, Camele, Ippolito NataleAbstract:This research was carried out to study in vitro antibacterial activity of 4 strains of Burkholderia gladioli pv. agaricicola (Bga) against G+ve Bacillus megaterium and Gve Escherichia coli, haemolytic activity against the cell membrane of erythrocytes, the production of extracellular hydrolytic enzymes and finally, the pathogenicity against Agaricus bisporus flesh blocks. Chemical structure of bioactive substances of the most bioactive strain (ICMP 11096) was established using gas chromatography–mass spectrometry (GC–MS). All the studied Bga strains inhibited the growth of the two tested bacteria although some growing substrates negatively influenced the antimicrobial substance production. The same Bga strains showed highly haemolytic activity and were able to produce 3 hydrolytic enzymes, i.e. chitinase, glucanase and protease. In pathogenicity assays, the considered Bga strains resulted virulent for A. bisporus. The GC–MS for compounds from Bga ICMP 11096 were compatible with the structure of two bioactive fatty acids identified as methyl stearate and ethanol 2-butoxy phosphate with mass spectrum m/e 298 and 398, respectively
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An attempt of biocontrol the tomato-wilt disease caused by Verticillium dahliae using Burkholderia gladioli pv. agaricicola and its bioactive secondary metabolites
'PAGEPress Publications', 2017Co-Authors: Elshafie, Hazem S., Bufo, Sabino Aurelio, Shimaa Sakr, Camele, Ippolito NataleAbstract:new microbial natural biocides such as microbial secondary metabolites to reduce the environmental pollution due to the excessive use of synthetic pesticides. Verticillium wilt, caused by the soil-borne Verticillium dahliae, is a widespread disease in tomato growing in many parts of the world. Burkholderia gladioli pv. agaricicola produces some antimicrobial substances and extracellular hydrolytic enzymes which exhibited promising antimicrobial activity towards several phytopathogens. The aims of the current research are to assess in vitro fungicidal effect of 4 strains of B. gladioli pv. agaricicola (ICMP11096, 11097, 12220 and 12322) against V. dahliae using culture or cell-free culture filtrate. In situ assay was performed to evaluate the biocontrol effect of the most efficient bacterial strain on wilt disease caused by V. dahliae in tomato plants. Results demonstrated that the studied bacterial strain ICMP12322 exerted the highest in vitro antifungal activity against V. dahliae which correlated with its ability to produce extracellular hydrolytic enzymes. Furthermore, in situ results showed that the selected bacterial strain significantly minimized the disease incidence
H S Elshafie - One of the best experts on this subject based on the ideXlab platform.
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An attempt of biocontrol the tomato-wilt disease caused by Verticillium dahliae using Burkholderia gladioli pv.agaricicola and its bioactive secondary metabolites
International Journal of Plant Biology, 2017Co-Authors: H S Elshafie, Sabino Aurelio Bufo, Shimaa H. Sakr, Ippolito Natale CameleAbstract:There is a great interest in discovering new microbial natural biocides such as microbial secondary metabolites to reduce the environmental pollution due to the excessive use of synthetic pesticides. Verticillium wilt, caused by the soil-borne Verticillium dahliae, is a widespread disease in tomato growing in many parts of the world. Burkholderia gladioli pv. agaricicola produces some antimicrobial substances and extracellular hydrolytic enzymes which exhibited promising antimicrobial activity towards several phytopathogens. The aims of the current research are to assess in vitro fungicidal effect of 4 strains of B. gladioli pv. agaricicola (ICMP11096, 11097, 12220 and 12322) against V. dahliae using culture or cell-free culture filtrate. In situ assay was performed to evaluate the biocontrol effect of the most efficient bacterial strain on wilt disease caused by V. dahliae in tomato plants. Results demonstrated that the studied bacterial strain ICMP12322 exerted the highest in vitro antifungal activity against V. dahliae which correlated with its ability to produce extracellular hydrolytic enzymes. Furthermore, in situ results showed that the selected bacterial strain significantly minimized the disease incidence.
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In vitro study of biological activity of four strains of Burkholderia gladioli pv. agaricicola and identification of their bioactive metabolites using GC-MS.
Saudi Journal of Biological Sciences, 2017Co-Authors: H S Elshafie, R Racioppi, Sabino Aurelio Bufo, Ippolito Natale CameleAbstract:This research was carried out to study in vitro antibacterial activity of 4 strains of Burkholderia gladioli pv. agaricicola (Bga) against G+ve Bacillus megaterium and G−ve Escherichia coli, haemolytic activity against the cell membrane of erythrocytes, the production of extracellular hydrolytic enzymes and finally, the pathogenicity against Agaricus bisporus flesh blocks. Chemical structure of bioactive substances of the most bioactive strain (ICMP 11096) was established using gas chromatography–mass spectrometry (GC–MS). All the studied Bga strains inhibited the growth of the two tested bacteria although some growing substrates negatively influenced the antimicrobial substance production. The same Bga strains showed highly haemolytic activity and were able to produce 3 hydrolytic enzymes, i.e. chitinase, glucanase and protease. In pathogenicity assays, the considered Bga strains resulted virulent for A. bisporus. The GC–MS for compounds from Bga ICMP 11096 were compatible with the structure of two bioactive fatty acids identified as methyl stearate and ethanol 2-butoxy phosphate with mass spectrum m/e 298 and 398, respectively.
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biochemical characterization of volatile secondary metabolites produced by Burkholderia gladioli pv agaricicola
International Journal of Drug Discovery, 2013Co-Authors: H S Elshafie, R RacioppiAbstract:Numerous species in the genus Burkholderia have interesting properties for potential industrial applications including production of antibiotics, biosurfactants, bioplastics and degradation of environmental contaminants. The aims of this study were to determine the antifungal activity of volatile secondary metabolites produced by four strains of Burkholderia gladioli pv. agaricicola (Bga) against the two phytopathogen- ic fungi Fusarium oxysporum and Rhizoctonia solani and to characterize biochemically the volatile organic compounds (VOCs) produced by the most bioactive Bga strain ICMP11096 tested in this study using Gas Chromatography-Mass Spectrometry. The studied strains showed antifungal activity against the tested phytopathogenic fungi through production of volatile bioactive metabolites. The biochemical characteriza- tion of VOCs of Bga ICMP11096 has detected two bioactive volatile compounds. The first one was a liquid hydrocarbon cyclic terpene and was identified as cyclohexene 1-methyl-4-(1-methylethenyl) and commonly considered one of the more frequent d- isomers of limonene. The second one was identified as 4-flavanone (4H-1-Benzopyran-4-one, 2, 3-dihydro-2-phenyl). The two produced VOCs could be the main re- sponsible for the antifungal activity.
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In Vitro Antifungal Activity of Burkholderia gladioli pv. agaricicola against Some Phytopathogenic Fungi
International Journal of Molecular Sciences, 2012Co-Authors: H S Elshafie, Nicola Sante Iacobellis, R Racioppi, Ippolito Natale Camele, Laura Scrano, Sabino Aurelio BufoAbstract:The trend to search novel microbial natural biocides has recently been increasing in order to avoid the environmental pollution from use of synthetic pesticides. Among these novel natural biocides are the bioactive secondary metabolites of Burkholderia gladioli pv. agaricicola (Bga). The aim of this study is to determine antifungal activity of Bga strains against some phytopathogenic fungi. The fungicidal tests were carried out using cultures and cell-free culture filtrates against Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. Results demonstrated that all tested strains exert antifungal activity against all studied fungi by producing diffusible metabolites which are correlated with their ability to produce extracellular hydrolytic enzymes. All strains significantly reduced the growth of studied fungi and the bacterial cells were more bioactive than bacterial filtrates. All tested Bulkholderia strains produced volatile organic compounds (VOCs), which inhibited the fungal growth and reduced the growth rate of Fusarium oxysporum and Rhizoctonia solani. GC/MS analysis of VOCs emitted by strain Bga 11096 indicated the presence of a compound that was identified as 1-methyl-4-(1-methylethenyl)-cyclohexene, a liquid hydrocarbon classified as cyclic terpene. This compound could be responsible for the antifungal activity, which is also in agreement with the work of other authors.
Yousef Dashti - One of the best experts on this subject based on the ideXlab platform.
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Genomics-driven discovery of a novel glutarimide antibiotic from Burkholderia gladioli reveals an unusual polyketide synthase chain release mechanism.
Angewandte Chemie International Edition, 2020Co-Authors: Gregory L. Challis, Matthew Jenner, Joleen Masschelein, Isolda Romero-canelón, Yousef Dashti, Ioanna T. Nakou, Eshwar MahenthiralingamAbstract:A gene cluster encoding a cryptic trans -acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters enabled gladiostatin, the metabolic product of the PKS, to be identified. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, indicating it plays a key role in polyketide chain release and butenolide formation.
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genomics driven discovery of a novel glutarimide antibiotic from Burkholderia gladioli reveals an unusual polyketide synthase chain release mechanism
Angewandte Chemie, 2020Co-Authors: Ioanna T. Nakou, Matthew Jenner, Joleen Masschelein, Yousef Dashti, Eshwar Mahenthiralingam, Gregory L. Challis, Isolda RomerocanelonAbstract:A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.
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Discovery and biosynthesis of gladiochelins: unusual lipodepsipeptide siderophores from Burkholderia gladioli
2020Co-Authors: Yousef Dashti, Ioanna T. Nakou, Alex J. Mullins, Gordon Webster, Xinyun Jian, Eshwar Mahenthiralingam, Gregory L. ChallisAbstract:Abstract Burkholderia is a genus of diverse Gram-negative bacteria that includes several opportunistic pathogens. Siderophores, which transport iron from the environment into microbial cells, are important virulence factors in most pathogenic Burkholderia species. However, it is widely believed that Burkholderia gladioli, which can infect the lungs of cystic fibrosis (CF) sufferers, does not produce siderophores. B. gladioli BCC0238, isolated from the lung of a CF patient, produces two novel metabolites in a minimal medium containing glycerol and ribose as carbon sources. HPLC purification, followed by detailed spectroscopic analyses, identified these metabolites as unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The absolute configurations of the amino acid residues in the two metabolites was elucidated using Marfey’s method and the gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of most B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S (CAS) assay showed the metabolites bind ferric iron and that this supresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes. Together, these observations suggest that these metabolites likely function as siderophores in B. gladioli.
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Kill and cure: genomic phylogeny and bioactivity of a diverse collection of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles
2020Co-Authors: Cerith Jones, Theodore Spilker, Matthew Jenner, Matthew J. Bull, Yousef Dashti, Alex J. Mullins, Gordon Webster, Julian Parkhill, Thomas R. Connor, John J. LipumaAbstract:Burkholderia gladioli is one of few bacteria with a broad ecology spanning disease in humans, animals, and plants, and encompassing beneficial interactions with multiple eukaryotic hosts. It is a plant pathogen, a bongkrekic acid toxin producing food-poisoning agent, and a lung pathogen in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the ecological diversity and specialized metabolite biosynthesis of 206 B. gladioli strains, phylogenomically defining 5 evolutionary clades. Historical disease pathovars (pv) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were phylogenetically distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable. Soft-rot disease and CF infection pathogenicity traits were conserved across all pathovars. Biosynthetic gene clusters for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade specific. Strikingly, 13% of CF-infection strains characterised (n=194) were bongkrekic acid toxin positive, uniquely linking this food-poisoning risk factor to chronic lung disease. Toxin production was suppressed by exposing strains to the antibiotic trimethoprim, providing a potential therapeutic strategy to minimise poisoning risk in CF.
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An unusual Burkholderia gladioli double chain-initiating nonribosomal peptide synthetase assembles ‘fungal’ icosalide antibiotics
'Royal Society of Chemistry (RSC)', 2019Co-Authors: Jenner Matthew, Jian Xinyun, Joleen Masschelein, Yousef Dashti, Hobson Christian, Roberts, Douglas M., Jones Cerith, Harris Simon, Parkhill Julian, Raja, Huzefa A.Abstract:Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 – a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly
