Mechanisms of polysaccharide degradation in particle-associated microbial communities

海洋颗粒中的微生物过程是海洋碳循环的核心，因为它们决定了浮游植物产生的碳的命运。颗粒相关（PA）细菌降解复杂的多糖，从而阻止它们成为海底的碳汇。颗粒的很大一部分是真核微生物，如活的或死的浮游植物。在B3中，我们利用元基因组学和元蛋白质组学技术研究天然浮游植物华，并结合培养中PA菌株的靶向分离和生理表征来解决这些颗粒的复杂性。我们开发了一种新的宏蛋白质组学管道来分析颗粒分数，并将其应用于2018年的浮游植物繁殖，揭示了PA生活方式的独特适应性。通过基于rRNA基因扩增子测序的共现网络分析，我们揭示了与含有硅藻或鞭毛藻的颗粒相关的不同细菌群落。在子项目B1中，我们发现这些细菌的宏基因组组装基因组反映了特定的藻-细菌相互作用。在长读元基因组测序的基础上，我们进一步能够从颗粒中组装真核生物基因组，这为解决浮游植物与细菌在多糖降解中的相互作用开辟了巨大的潜力。沉降锥的引入进一步使我们能够识别具有颗粒相关或附着生活方式的模式细菌。此外，我们建立了PA细菌的收集。其中，选择Muricauda和Maribacter菌株作为模型PA细菌，对不同多糖培养的细胞进行蛋白质组学分析。蛋白质组学分析确定了具有未知底物特异性的新型糖基水解酶的表达。在Maribacter中，作为底物的天然微藻细胞壁诱导了与阿拉伯半乳聚糖高度相似的蛋白质组模式。在子项目B3的第三阶段，我们通过引入真核生物基因组学专家新PI，扩大了颗粒微生物组的探索，特别是它们的真核成分。通过从宏基因组数据中组装和注释真核生物基因组，我们将突破目前解释海洋颗粒复杂性的界限。再加上对2020年春季开花的前所未有的高分辨率元蛋白质组学分析，以及更复杂的细菌-浮游植物共生网络分析，我们将能够阐明浮游植物-细菌相互作用的真核和细菌方面及其对降解和多糖合成的影响。此外，我们计划对典型海洋颗粒底物转化过程中诱导的细菌酶进行生化表征，扩大我们对海洋多糖活性酶多样性的理解。

Microbial processes in marine particles are central to carbon cycling in the oceans, as they determine the fate of phytoplankton-produced carbon. Particle-associated (PA) bacteria degrade complex polysaccharides, thereby preventing them from becoming a carbon sink on the ocean floor. A large fraction of particles are eukaryotic microorganisms such as living or dead phytoplankton. In B3, we tackle the complexity of these particles using metagenomic and metaproteomic techniques on natural phytoplankton blooms in combination with targeted isolation and physiological characterization of PA bacterial strains in culture. We developed a novel metaproteomics pipeline to analyze the particle fraction and applied this to the phytoplankton bloom in 2018, uncovering unique adaptations of the PA lifestyle. With co-occurrence network analyses based on rRNA gene amplicon sequencing, we revealed distinct bacterial communities associated with particles containing either diatoms or dinoflagellates. With subproject B1, we showed that metagenome-assembled genomes of these bacteria reflect specific algal-bacterial interactions. On the basis of long-read metagenome sequencing, we were further able to assemble eukaryotic genomes from particles, which opens up enormous potential for addressing interactions between phytoplankton and bacteria in polysaccharide degradation. The introduction of sedimentation cones further enabled us to identify model bacteria with a particle-associated or -attached lifestyle. Further, we established a collection of PA bacteria. Of these, Muricauda and Maribacter strains were selected as model PA bacteria for the proteomic analysis of cells grown on different polysaccharides. Proteomic analyses identified the expression of novel glycosyl hydrolases with unknown substrate specificity. In Maribacter, natural microalgal cell walls as a substrate induced highly similar proteome patterns as arabinogalactan. In the third phase of subproject B3, we expand the exploration of particle microbiomes and, in particular, of their eukaryotic component by including new PI, an expert in eukaryote genomics. By assembling and annotating eukaryotic genomes from metagenomic data, we will push the current boundaries for interpreting the complexity of marine particles. Coupled with an unprecedented high-resolution metaproteomic analysis of the spring bloom of 2020, as well as more sophisticated bacterial-phytoplankton co-occurrence network analysis, we will be able to shed light on both the eukaryotic and bacterial sides of phytoplankton-bacterial interactions and their impact on degradation as well as synthesis of polysaccharides. Furthermore, we plan to biochemically characterize bacterial enzymes which are induced during transformations of substrates typical of marine particles, expanding our understanding of the diversity of marine polysaccharide-active enzymes.