In situ mechanisms of polysaccharide degradation of key bacteroidetal genera in spring algae blooms

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In this subproject, we investigate in situ mechanisms of polysaccharide degradation by key flavobacterial species in spring diatom blooms in the North Sea at Helgoland. The combination of proteomic methods with fluorescence in situ hybridization (FISH) enables us to determine the diversity and abundance of flavobacterial clades, and to discover which carbohydrate-degrading enzymes they express during the course of the bloom. In the previous POMPU funding period and in a collaborative effort, we determined the microbial community composition of the spring phytoplankton bloom 2020. This data was pivotal to put the focus on key time points for the spring bloom for in-depth analyses for all other subprojects. As observed in previous blooms, in 2020 strong successional patterns of distinct, mostly flavobacterial clades were discernable. These clades recurrently appear in many of the spring blooms examined to date and are affiliated to the genera Aurantivirga, Polaribacter and Candidatus Prosiliicoccus. By continuous method development of the proteomic tools exceptionally high protein detection rates (>27,000) enabled us to follow distinct carbohydrate-active enzymes (CAZyme) and helped to identify the main degraders of laminarin and fucose-containing sulfated polysaccharides throughout the bloom in 2016. For the diatom bloom in 2020, about 33,000 protein groups of the bacterial fractions could be identified so far. Furthermore, we have developed a method to identify and localize key bacteria in plankton samples using polynucleotide probes specific for genes encoding carbohydrate-active enzymes. In an alternative approach, we have used fluorescently labeled polysaccharides to stain bacteria that take up the respective glycan. By a combination of FISH, flow cytometry and subsequent sequencing, we could show that a previously unknown verrucomicrobial clade, the Pedosphaeraceae, was able to take up massive amounts of fluorescent laminarin. For proteomic analyses we realized many methodological improvements, which enabled us to gain insights into the mechanisms of polysaccharide degradation in situ. Several protocols were tested on cultured Polaribacter strain KT25b, allowing us to reduce the input material required for proteomic analyses to as little as 100,000 cells. However, what is feasible for cultured Polaribacter still remains challenging for “wild catches” (environmental samples), which are both smaller in cell size and more diverse. In the third funding period, we will continue to focus on proteomics and the visualization of polysaccharide degradation in situ. Specifically, we will supplement the already available proteomics methods with new targeted proteome techniques. Our in situ analyses include new aspect, such as the visualization of specific traits with polynucleotide probes focusing on CAZyme-coding genes and high-resolution fluorescence microscopy.