Physiological proteomics of relevant enzymes and transporters in marine polysaccharide-degrading Bacteroidetes

A1子项目旨在描述专门降解多糖的海洋拟杆菌的生理学和适应性。我们将从功能上分析决定浮游植物水华期间关键细菌分支成功的分子机制，以及它们在多糖利用中的底物专一性。各自的酶在特定的基因组区域编码，即所谓的多糖利用基因座(Puls)。我们使用可培养关键细菌在特定底物条件下的比较定量亚蛋白质组学分析来推断选定的Puls及其编码的CAZyme和转运蛋白的特定功能。该项目的重点是细菌分离物，它们已被确定为与北海春季浮游植物水华有关。实验室培养结果与环境数据的相关性是确定关键细菌的生态位及其在自然界海洋多糖周转中的相互作用的关键前提。在第一和第二资助阶段，子项目A1集中在α-和β-葡聚糖特定的降解过程，并确定了负责利用这些丰富的海洋糖的关键细菌、PUL类型和蛋白质功能。此外，我们还支持对A2、A3、A4和B1中的岩藻糖胶、岩藻糖胶、木聚糖和甘露聚糖的降解过程进行详细的功能分析。与该联盟的合作伙伴一起，我们能够确定海洋拟杆菌菌株对这些藻类多糖的主要降解途径以及新的CAZyme功能。在第三个POMPU阶段，我们的目标是完成我们对海洋拟杆菌中海带蛋白特异性蛋白的细胞定位和推测的功能连锁的详细研究。我们假设，糖结合、降解和运输蛋白质的严格有序的蛋白质机制是一种关键的分子适应，它促进了多糖在高度扩散的海洋环境中的降解。由于海带多糖是海洋栖息地中含量最丰富的多糖，我们将继续对在硅藻驱动的水华期间在赫尔戈兰分离株和环境样品中检测到的不同类型的海带多糖响应脉冲进行功能分析。在POMPU的最后阶段，我们将完成对潜在调控机制的详细和系统的分析，这些机制决定了底物特定的Puls的分级表达，从而控制了浮游植物水华期间天然海洋多糖的降解。我们将进一步完成对一种新的调节剂类型的海带蛋白脉冲的功能和结构分析(在第二个POMPU阶段发现)，从而完成对这些重要的海洋PUL功能的研究。

Subproject A1 aims to characterize the physiology and adaptation of marine Bacteroidetes that specialize in polysaccharide degradation. We will functionally analyze molecular mechanisms which determine the success of key bacterial clades during phytoplankton blooms and their substrate specificity in polysaccharide utilization. The respective enzymes are encoded in dedicated genomic regions, so-called polysaccharide utilization loci (PULs). We use comparative quantitative subproteomic analyses of cultivable key bacteria under defined substrate conditions to deduce specific functions of selected PULs and their encoded CAZymes and transporters. In the focus of this project are bacterial isolates, which have been identified as relevant during spring phytoplankton blooms in the North Sea. The correlation of results obtained from defined laboratory cultivations on the one side and from environmental data on the other side is a crucial prerequisite to define ecological niches of key bacteria and their interaction in marine polysaccharide turnover in nature.In the first and second funding phase, subproject A1 focused on alpha- and beta-glucan specific degradation processes and determined key bacteria, PUL types and protein functions responsible for the utilization of these abundant marine sugars. Furthermore, we supported the detailed functional analyses of ulvan-, fucoidan-, xylan- and mannan- degradation processes in A2, A3, A4 and B1. Together with the consortium partners, we were able to determine major degradation pathways as well as new CAZyme functions of marine Bacteroidetes strains for these algal polysaccharides. During the third POMPU phase, we aim to finalize our detailed investigation of the cellular localization and putative functional interlinkages of laminarin-specific proteins in marine Bacteroidetes. We hypothesize that a strictly ordered protein machinery of sugar-binding, -degrading and -transporting proteins is a crucial molecular adaptation, which facilitates polysaccharide degradation in the highly diffusible marine environment. Since laminarin is the most abundant polysaccharide in marine habitats, we will continue to functionally analyze different types of laminarin-responsive PULs detectable in our Helgoland isolates and environmental samples during diatom-driven blooms. In this concluding POMPU phase, we will complete our detailed and systematic analyses of potential regulatory mechanisms, which determine a hierarchical expression of substrate-specific PULs and thus a controlled degradation of natural marine polysaccharides during phytoplankton blooms. We will furthermore finalize our functional and structural analyses of a new regulator type of laminarin PULs (discovered in the second POMPU phase), thus completing our investigation of these important marine PUL functions.