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

在这个子项目中，我们调查在北海Helgoland春季硅藻水华中关键黄细菌物种的多糖降解的原位机制。蛋白质组学方法与荧光原位杂交（FISH）相结合，使我们能够确定的多样性和丰富的flavobacterial分支，并发现他们在开花过程中表达的碳水化合物降解酶。在之前的POMPU资助期间和合作努力中，我们确定了2020年春季浮游植物水华的微生物群落组成。这些数据对于将重点放在春季开花的关键时间点，以便对所有其他子项目进行深入分析至关重要。正如在以前的水华中所观察到的那样，在2020年，可以看出明显的、主要是黄细菌分支的强烈演替模式。这些分支经常出现在许多春季开花检查日期，并隶属于属Aurantivirga，Polaribacter和Prosiliicoccus。通过蛋白质组学工具的持续方法开发，极高的蛋白质检测率（> 27，000）使我们能够跟踪不同的碳水化合物活性酶（CAZyme），并帮助确定2016年整个水华期间海带多糖和含岩藻糖硫酸多糖的主要降解剂。对于2020年的硅藻水华，到目前为止可以鉴定出约33，000个细菌组分的蛋白质组。此外，我们已经开发了一种方法来识别和定位浮游生物样品中的关键细菌，使用多核苷酸探针的编码碳水化合物活性酶的基因。在另一种方法中，我们使用荧光标记的多糖来染色吸收相应聚糖的细菌。通过FISH，流式细胞术和随后的测序相结合，我们可以表明，一个以前未知的疣状微生物分支，Pedosphaeraceae，能够采取大量的荧光海带多糖。对于蛋白质组学分析，我们实现了许多方法上的改进，这使我们能够深入了解多糖原位降解的机制。在培养的极化杆菌菌株KT 25 b上测试了几种方案，使我们能够将蛋白质组学分析所需的输入材料减少到100，000个细胞。然而，对于培养的极化杆菌来说可行的方法对于“野生捕获物”（环境样品）仍然具有挑战性，野生捕获物的细胞尺寸更小，而且更多样化。 在第三个资助期，我们将继续专注于蛋白质组学和多糖原位降解的可视化。具体来说，我们将补充现有的蛋白质组学方法与新的靶向蛋白质组技术。我们的原位分析包括新的方面，如特定性状的可视化与多核苷酸探针集中在CAZyme编码基因和高分辨率荧光显微镜。

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.