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，从而防止它们成为海底的碳汇。很大一部分颗粒是真核微生物，如活的或死的浮游植物。在B3中，我们使用对自然浮游植物水华的元基因组和元蛋白质组技术，结合培养中PA细菌菌株的有针对性的分离和生理特性，来解决这些颗粒的复杂性。我们开发了一种新的元蛋白质组学管道来分析颗粒组分，并将其应用于2018年浮游植物的水华，揭示了PA生活方式的独特适应。通过基于rRNA基因扩增子序列的共生网络分析，我们揭示了与含有硅藻或甲藻的颗粒相关的独特的细菌群落。通过B1子项目，我们证明了这些细菌的元基因组组装反映了特定的藻类-细菌相互作用。在长时间阅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基因组，这为解决浮游植物和细菌之间在多糖降解过程中的相互作用开辟了巨大的潜力。沉淀锥的引入进一步使我们能够识别具有与颗粒相关的或附着的生活方式的模型细菌。此外，我们建立了PA细菌的集合。其中，Muricauda和Maribacter菌株被选为模式PA细菌，用于对生长在不同多糖上的细胞进行蛋白质组学分析。蛋白质组学分析证实了新的糖基水解酶的表达，底物特异性未知。在Maribacter中，以天然微藻细胞壁为底物，诱导出与阿拉伯半乳糖高度相似的蛋白质组模式。在B3子项目的第三阶段，我们通过包括真核生物基因组学专家新的PI，扩大了对粒子微生物组的探索，特别是对它们的真核成分的探索。通过从元基因组数据中组装和注释真核基因组，我们将突破当前的界限，解释海洋颗粒的复杂性。结合对2020年春季水华的前所未有的高分辨率元蛋白质组分析，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，我们计划对海洋颗粒典型底物转化过程中诱导的细菌酶进行生化表征，以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.