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Characterization and study of the regulation of mupirochelin, a new Pseudomonas siderophore with anti-oomycete activity

par Grosse, Camille
Président du jury Meyer, Franck
Promoteur Wintjens, René
Co-Promoteur Matthijs, Sandra
Publication Non publié, 2023-09-25

Thèse de doctorat

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Résumé :

Globisporangium ultimum is an oomycetal pathogen causing damping-off on over 300 different plant hosts. Currently, as for many phytopathogens, its control relies in the use of chemicals with negative impact on health and ecosystems. Therefore, many biocontrol strategies are under investigation to reduce the use of fungicides. In this study, the soil bacterium Pseudomonas sp. NCIMB 10586 demonstrates a strong iron-repressed in vitro antagonism against G. ultimum MUCL 38045 caused by the secretion of a novel non-ribosomal peptide synthetase (NRPS) siderophore named mupirochelin. Its putative structure obtained by MS/MS analysis bears similarities to other siderophores and bioactive compounds. Besides mupirochelin, we observed the production of a third and novel NRPS-independent siderophore (NIS), here termed triabactin. The iron-responsive transcriptional repression of the two newly identified siderophore gene clusters corroborates their role as iron scavengers. However, their respective contributions to the strain fitness are dissimilar. Bacterial growth in iron-deprived conditions is greatly supported by pyoverdine production and, to a lesser extent, by triabactin. On the contrary, mupirochelin does not contribute to the strain fitness under the studied conditions. Besides a possible repression induced by the ferric uptake regulator (Fur), the expression of mupirochelin gene cluster has been shown to be activated by the AraC-like transcriptional regulator MchR. RT-qPCR results suggest that MchR activates mupirochelin biosynthesis when it binds the ferri-mupirochelin chelate. Therefore, the regulation of mupirochelin pathway is tightly controlled by iron availability. The predictive structure of MchR generated with Alphafold2 indicates the presence of a N-terminal effector-binding domain containing a large hydrophobic cavity buried in its centre and presumed to accommodate mupirochelin. Purification of a recombinant MBP-MchR fusion protein was achieved to characterize its mechanism of regulation. The binding of the ferri-mupirochelin chelate to MchR was observed by LC-MS analysis and corroborates our previous observations. Although EMSA analysis suggests that apo MchR does not bind to the DNA, TSA analysis indicates a possible interaction of the protein with some of the putative MchR-box predicted by sequence analysis. Therefore, further work is needed to characterize MchR mechanism of regulation. Altogether, we have demonstrated here that besides pyoverdine, Pseudomonas sp. NCIMB 10586 produces two newly identified siderophores, namely mupirochelin, a weak siderophore with strong antagonism activity against G. ultimum, and the potent siderophore triabactin. In conclusion, the knowledge gained from this work contributes to the understanding of mupirochelin physiological role in a bacterial strain producing three chemically different siderophores.