Studies of culture conditions on the production of metabolites by lichen-associated bacteria
Résumé
Lichens are commonly described as a mutualistic symbiosis between fungi and alga. However, recent studies revealed the diversity in bacteria inhabiting lichens, suggesting that they host assemblages from several bacteria phyla such as Actinobaceria, Proteobacteria, and Firmicutes. These microorganisms are promising sources of structurally diverse and potent bioactive compounds. As part of our search for novel sources of bioactive natural products, the bacterial community of the lice Peltigera hymeniana from Brittany (France) was studied. Different microorganisms were isolated; among them, three bacteria of the genus Bacillus were retaned for their promising production of cytotoxic and/or antibacterial compounds: Lysinibacillus fusiformis, Bacillus mycoides, and Bacillus weihenstephanensis. Bacillus species are known for their synthesis of secondary metabolites including bioactive isocoumarns, lipopetides, macrolactones and polypeptides.
To improve the production yields and the diversity of metabolites of therapeutic interest, our strategy was based on optimization of bacterial growth conditions. First, we studied the influence of nutrients and culture length on the metabolite production. Different media were used as various nutrients carriers. In addition, influence of incubation periods was estimated by comparing cultures stopped after 1, 4, and 7 days past reaching stationary phase. Extractions of both culture media and cell pellet were carried out leading to complex extracts. Our studies combined (i) LC-MS analyses and data processing methods for visualizing sample profile and diversity, (ii) antibacterial and cytotoxic tests as bio-guided assays, and (iii) semi-preparative HPLC, NMR data and exact mass analyses for the elucidation of chemical composition of bacterial extracts. This approach allowed to assess the diversity and redundancy of the compounds depending on culture conditions and led to the production of potential therapeutic agents.