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

子项目A1旨在描述海洋拟杆菌门（Bacteroidetes）的生理特征和适应性，这些拟杆菌门专门降解多糖。我们将从功能上分析决定浮游植物繁殖过程中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特异性。各自的酶编码在专门的基因组区域，即所谓的多糖利用位点（PULs）。我们在确定的底物条件下对可培养的关键细菌进行比较定量的亚蛋白质组学分析，以推断选定的PULs及其编码的CAZymes和转运体的特定功能。这个项目的重点是分离的细菌，它们已经被确定为与北海春季浮游植物大量繁殖有关。将实验室培养的结果与环境数据的结果相关联，是确定关键细菌的生态位及其在自然界中海洋多糖转化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侧重于α -葡聚糖和β -葡聚糖特异性降解过程，并确定了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，我们支持了A2， A3， A4和B1中ulvan-，岩藻聚糖-，木聚糖-和甘露聚糖-降解过程的详细功能分析。与合作伙伴一起，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门菌株的新CAZyme功能。在POMPU的第三阶段，我们的目标是完成我们对海洋拟杆菌门中层状蛋白特异性蛋白的细胞定位和推测的功能相互联系的详细研究。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个关键的分子适应，它促进了在高度扩散的海洋环境中多糖的降解。由于藻胶蛋白是海洋栖息地中最丰富的多糖，我们将继续对Helgoland分离物和环境样品中检测到的不同类型的藻胶蛋白敏感的PULs进行功能分析。在最后的POMPU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异性PULs的层次表达，从而在浮游植物繁殖期间控制天然海洋多糖的降解。我们将进一步完成对一种新的调节型层状蛋白PULs（在POMPU的第二个阶段发现）的功能和结构分析，从而完成我们对这些重要的海洋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.