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

子项目A1的目的是描述专门降解多糖的海洋拟杆菌的生理和适应。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及其在多糖利用中的底物特异性。相应的酶在专用基因组区域，即所谓的多糖利用基因座（普尔斯）中编码。我们使用比较定量亚蛋白质组学分析的可培养的关键细菌在确定的底物条件下推断特定功能的选择普尔斯和他们的编码的CAZyme和转运。该项目的重点是细菌分离株，已被确定为与北海春季浮游植物水华有关。一方面从确定的实验室培养获得的结果与另一方面从环境数据获得的结果之间的关联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先决条件，在第一和第二资助阶段，子项目A1侧重于α-和β-葡聚糖的特定降解过程，并确定了关键细菌，PUL类型和蛋白质功能负责利用这些丰富的海洋糖。此外，我们支持详细的功能分析的石莼多糖，岩藻依聚糖，木聚糖和甘露聚糖降解过程中A2，A3，A4和B1。与财团合作伙伴一起，我们能够确定这些藻类多糖的主要降解途径以及海洋拟杆菌属菌株的新CAZyme功能。在第三POMPU阶段，我们的目标是完成我们的详细调查的细胞定位和推定的功能相互联系的海带蛋白特异性蛋白在海洋拟杆菌。我们假设，严格有序的糖结合，降解和运输蛋白质的蛋白质机械是一个至关重要的分子适应，这有利于多糖降解在高度扩散的海洋环境。由于海带多糖是海洋栖息地中最丰富的多糖，我们将继续功能分析不同类型的海带多糖响应普尔斯检测在我们的Helgoland分离物和环境样品中的藻类驱动的水华。在这一结论POMPU阶段，我们将完成我们的详细和系统的分析，潜在的监管机制，这决定了一个分层表达的基板特定的普尔斯，从而控制降解天然海洋多糖在浮游植物水华。我们将进一步完成我们的功能和结构分析的一种新的调节器类型的海带多糖普尔斯（发现在第二POMPU阶段），从而完成我们的调查，这些重要的海洋PUL功能。

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.