Project area B: Bacterial adaptation strategies to the oak phyllosphere
In this project area, bacterial adaptation to the phyllosphere is investigated. It focuses on a molecular-mechanistic characterization of relevant metabolic pathways and their environment-dependent regulation in cultured isolates of the bacterial leaf microbiota, in vitro as well as in situ in mesocosms and on oaks in the forest ecosystem.
Project B1: Biosynthesis of secondary metabolites by leaf microbiota
Helge Bode, Robert Junker
Members of the oak leaf microbiota encoding biosynthetic gene clusters (BGCs) involved in the production of natural products like polyketides, terpenes or peptides are analyzed by mass spectrometry (HPLC/MS and MALDI/MS). BGCs will be activated using global and specific regulators as well as direct BGC activation via promoter-exchange approaches in order to identify, isolate and elucidate the structure (by mass spectrometry and nuclear magnetic resonance) of the respective natural products and to correlate their production with ‘on-leaf production’ using imaging mass spectrometry. Mutants with a modified production of natural products also found on leaves will be analyzed in synthetic leaf microbiota communities in order to understand the contribution of these natural products for the microbial community.
Project B2: Carbon metabolism of bacterial leaf microbiota
The oak leaf microbiota are able to utilise different carbon sources from oak leaves, such as methanol and isoprene. In this project, we want to isolate bacteria capable of oxidising methanol and isoprene and then identify novel methanol dehydrogenases and isoprene monooxygenases. Genome, proteome/transcriptome and genome editing will be used to investigate the genes related to the isoprene degradation pathway and then help us understand how bacteria regulate isoprene oxidation. Furthermore, the abundance, diversity and composition of isoprene-degrading bacteria in different seasons of oak phyllosphere will be evaluated, providing a better understanding of the relationship between leaves, microbes and isoprene emissions and thus the impact of oak trees on the atomosphere and humans.
Project B3: Nitrogen fixation by diazotrophic bacteria of the leaf microbiota
Johannes Rebelein, Anke Becker
The oak leaf microbiota will be analyzed for the expression of nitrogenases. Nitrogenase activity assays will be established to quantify the fixation of N2 on the leaf. The molecular adaption of diazotrophic bacteria to the environment oak leaf will be characterized. Nitrogen fixation genes of selected bacteria will be engineered to influence the nitrogen fixation on the leaf and the impact will be evaluated.
Project B4: Resource-dependent regulation of bacterial primary and secondary metabolism
Sandra Schuller, Jan Schuller, Gert Bange
Bacterial primary and secondary metabolism is adapted to the diurnal cycles on the plant surface. In this project we want to investigate to what extent bacterial adaptation mechanisms play a role. One focus is on the metabolic regulation of methylotrophic bacteria living on the leaf surface, whose food supply strongly depends on the day and night metabolism of the plants. To study the protein signalling systems involved, we use classical protein biochemistry, minimal in vitro systems and structural biology methods, in particular cryoEM.
