Degradation of the mycotoxin fusaric acid in burkholderia ambifaria t16: genes and metabolic pathways involved

Fusaric acid (FA, 5-butylpyridine, 2-carboxylic acid) is a secondary metabolite produced by several Fusarium species, which is toxic for bacteria, plants, animals and humans. This mycotoxin contributes to the virulence of phytopathogenic Fusarium in several crops, causing important economic losses. Moreover, FA reduces survival and competition abilities of bacterial species able to antagonize Fusarium spp. due to its negative effects on viability and production of antibiotics effective against these fungi. Burkholderia ambifaria T16 is a bacterial strain isolated from the rhizosphere of barley that showed the interesting ability to degrade FA and detoxify this mycotoxin from barley seedlings. The genes and metabolic pathways involved in FA degradation have not been identified so far in any bacterial species. By screening of a transposon insertion library and proteomic analysis we were able to identify genes and metabolic pathways that would be involved in FA degradation. A functional 2-methylcitrate cycle (2-MCC), a carbon anaplerotic pathway widely distributed among bacteria and fungi where propionyl-CoA is converted to pyruvate and succinate, was shown to be essential for the growth of B. ambifaria T16 in the presence of FA. Propionyl-CoA and its derived catabolites are lethally toxic to cells when accumulate. For that reason, besides providing succinate and pyruvate, the 2-MCC also has a very important role in the detoxification of propionyl-CoA and its catabolites. The comparison of the proteomic profile of B. ambifaria T16 growing with FA or citrate as sole carbon sources showed that more than 50 enzymes were significantly overexpressed during growth with FA, including 2-MCC enzymes and enzymes that convert butyryl-CoA to propanoyl-CoA, suggesting that propanoyl-CoA is produced during FA degradation. Moreover, several proteins, including an AraC-type transcriptional regulator, a FMN-dependent two-component luciferase like monooxygenase (LLM) system, an amidohydrolase, two enoyl-CoA hydratases and a long-chain fatty acid ligase, encoded in the same gene cluster, were highly over-expressed during growth with FA (>10 fold up-regulation). In the last years, two-component LLMs were shown to catalyze the pyridine-ring cleavage of several N-heterocyclic compounds, suggesting that the mentioned gene cluster is a good candidate to be involved in the initial steps of FA degradation in B. ambifaria T16.Fil: Vinacour, Matias Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Forne, I.. Ludwig Maximilians Universitat; AlemaniaFil: Jung, K.. Ludwig Maximilians Universitat; AlemaniaFil: Imhof, A.. Ludwig Maximilians Universitat; AlemaniaFil: Ruiz, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaLVII SAIB Meeting; XVI SAMIGE MeetingCiudad Autonoma de Buenos AiresArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularSociedad Argentina de Microbiología Genera

Genomic insights into the potent antifungal activity of B. ambifaria T16

Burkholderia ambifaria T16 is a bacterial strain isolated from the rhizosphere of barley plants, which showed a potent antifungal activity against several Fusarium species. In this work, the construction of a mini-Tn5 insertional library in this strain allowed us the identification of several genes involved in the antifungal activity. In particular, several mutants with mini-Tn5 insertions located in a biosynthetic gene cluster (BGC) involved in the production of cyclic glycolipopeptides known as occidiofungins (Ocfs) were completely unable to inhibit the in vitro growth of F. oxysporum. Tandem mass spectrometry analysis of lipopeptide extracts obtained from culture supernatants of the wild-type strain allowed the identification of eight Ocfs structural variants, that were not detected in extracts of the mutants with insertions in the ocf gene cluster. These results indicate that the antifungal activity of B. ambifaria T16 mainly relies on Ocfs production. Moreover, an insertion in a BGC predicted to be involved in the biosynthesis of a membrane-associated lipopeptide designated AFC-BC11 reduced the antifungal activity of B. ambifaria T16 by approximately 50%. Accordingly, membrane extracts from the insertional mutant in the afc-BC11 cluster showed a marked reduction in the antifungal activity against several fungi. In contrast, the antifungal activity of the membrane fraction of mutants unable to produce Ocfs was not affected. Furthermore, a detailed bioinformatic analysis of the proteins encoded by the ocf and afc-BC11 gene clusters revealed that the biosynthesis of these lipopeptides would be initiated by fatty acyl-AMP ligase (FAAL) domain containing enzymes and continued by a hybrid non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) or a type III PKS, respectively. These results provide new insights into lipopeptide biosynthesis, which may be valuable in the field of novel antifungal compound design.Fil: Simonetti, Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Alvarez, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Feldman, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Vinacour, Matias Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Roberts, Irma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Ruiz, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentin

Genetic dissection of the degradation pathways for the mycotoxin fusaric acid in Burkholderia ambifaria T16

Fusaric acid (FA) is a mycotoxin produced by several Fusarium species. Burkholderia ambifaria T16 is a rhizosphere bacterium, able to use FA as sole nitrogen, carbon and energy source. By screening a transposon insertional library, combined with proteomic analysis, genes and enzymes involved in the microbial degradation of FA were identified for the first time. A functional 2-methylcitrate cycle, an anaplerotic pathway where propionyl-coenzyme A (CoA) is converted to pyruvate and succinate, was shown to be essential for growth in the presence of FA. The proteomic profile of B. ambifaria T16, showed that more than 50 enzymes (including those belonging to the 2-methylcitrate cycle, fatty acid metabolism, valine catabolism and flavin biosynthesis), were significantly more abundant when growing on FA than on citrate. Flavin mononucleotide (FMN)-dependent luciferases like monooxygenase (LLMs) are shown to catalyze the pyridine-ring cleavage reaction of several N-heterocyclic compounds. Deletion of a gene encoding a predicted LLM enzyme that was highly up-regulated during growth on FA, completely abolished the capability of B. ambifaria T16 to grow with this mycotoxin as sole nitrogen, carbon and energy source. Re-introduction of the wild type gene was able to restore growth. The mentioned gene is part of a gene cluster of unknown function that we termed fua, due to its probable role in fusaric acid catabolism. Our results suggest that the LLM encoded in the fua cluster catalyzes the pyridine-ring opening reaction during FA degradation, and that propionyl-CoA is one of the intermediates of FA catabolism in B. ambifaria T16.Fil: Vinacour, Matias Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Moiana, Mauro Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Forné, Ignasi. Ludwig Maximilians Universitat; AlemaniaFil: Jung, Kirsten. Ludwig Maximilians Universitat; AlemaniaFil: Bertea, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Calero Valdayo, Patricia M.. Technical University of Denmark; DinamarcaFil: Nikel, Pablo I.. Technical University of Denmark; DinamarcaFil: Imhof, Axel. Ludwig Maximilians Universitat; AlemaniaFil: Palumbo, Miranda Clara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Ruiz, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentin