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

在这个子项目中，我们研究了Helgoland北海春季硅藻华中关键黄杆菌种降解多糖的原位机制。蛋白质组学方法与荧光原位杂交（FISH）的结合使我们能够确定黄杆菌分支的多样性和丰度，并发现它们在开花过程中表达哪些碳水化合物降解酶。在之前的POMPU资助期和合作努力下，我们确定了2020年春季浮游植物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上，以便对所有其他子项目进行深入分析是至关重要的。正如在之前的花中观察到的那样，在2020年，可以看到明显的、主要是黄杆菌分支的强烈演替模式。到目前为止，这些分支在许多春季开花中反复出现，隶属于Aurantivirga属，Polaribacter属和Candidatus proiliicoccus属。通过蛋白质组学工具的持续方法开发，异常高的蛋白质检测率（bbb27,000）使我们能够跟踪不同的碳水化合物活性酶（CAZyme），并帮助确定了2016年整个水华期间层压蛋白和含焦点的硫酸盐多糖的主要降解剂。对于2020年的硅藻华，到目前为止，可以识别出大约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本中的关键细菌，使用特定于编码碳水化合物活性酶基因的多核苷酸探针。在另一种方法，我们已经使用荧光标记的多糖染色细菌，采取各自的聚糖。通过结合FISH、流式细胞术和随后的测序，我们可以证明一个以前未知的verrucomicromicals分支，Pedosphaeraceae，能够吸收大量的荧光层粘连蛋白。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的原位降解机制。在培养的极化杆菌菌株KT25b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“野生捕获物”（环境样本）来说，培养极化杆菌的可行性仍然是一个挑战，因为它们的细胞大小更小，种类也更多样化。在第三个资助期，我们将继续关注蛋白质组学和多糖原位降解的可视化。具体来说，我们将用新的靶向蛋白质组技术补充现有的蛋白质组学方法。我们的原位分析包括新的方面，如特异性状的可视化与多核苷酸探针聚焦于cazye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.