Comparative (meta-)genomics of marine polysaccharide-degrading bacteria with emphasis on CAZyme and PUL analyses in Bacteroidetes

分项目B1侧重于多糖降解细菌的比较（Meta）基因组学。海洋浮游植物大量繁殖是全球碳循环的关键事件。它们清除了大气中数十亿吨的二氧化碳，并引发了异养浮游细菌分支的连续大量繁殖，这些分支将大部分固定碳固定化。在POMPU之前，我们在北海岛黑尔戈兰岛（2009-2012）的四个春季开花期间研究了这种演替。使用一个集成的OMICS方法，我们发现，经常丰富的浮游细菌分支表现出显着不同的基板光谱。这是特别明显的基因频率和表达的碳水化合物活性酶（CAZymes）在拟杆菌门，表明生态位分区藻类多糖降解。在拟杆菌门中，编码多糖摄取和降解机制的基因通常被组织在称为“多糖利用位点”（普尔斯）的专用位点中，并且PUL分析可以潜在地预测目标多糖类。到目前为止，我们在POMPU中的作用是通过对（i）分离株和（ii）大小分级的微生物群落的比较基因组分析来研究海洋多糖利用。在第一个项目阶段，我们分析了（i）53个测序的北海拟杆菌菌株的PUL库和（ii）38个分类和分类的北海宏基因组的时间序列，以揭示0.2-3 μm（自由生活）部分中所有丰富细菌的PUL库，包括尚未培养的拟杆菌。在第二个项目阶段，我们将重点转移到（i）Helgoland春季开花期间Gammaproteobacteria的生态位（ii）自由生活与颗粒相关拟杆菌门的比较基因组学（3- 10 μm和>10 μm部分）在2018年春季水华期间，和（iii）不同游离-在2020年春季水华期间，使用宏基因组学、转录组学和蛋白质组学的组合，大幅提高了时间分辨率的活浮游细菌分支（A1，Z）。后两种方法生成了全面的数据集，其分析将导致对两种截然不同的水华的综合生态学解释。在POMPU扩展期间，我们希望完成对2018年和2020年水华的分析，并利用3- 10 μm粒径的颗粒附着细菌数据补充2020年的研究，特别是关于获得更高质量的MAG（宏基因组组装的基因组）和高时间分辨率的元转录组数据。这些数据还将为次级项目B3、B2和A1正在进行的一揽子工作提供基础。此外，我们将专注于尚未探索的多糖降解进化枝的详细研究，将糖测量（A4）与OMICS数据相结合，并协助基因FISH实验，以直观地将选定的普尔斯分配给自由生活或颗粒附着的细菌（B2）。

Subproject B1 focuses on comparative (meta-)genomics of polysaccharide-degrading bacteria. Marine phytoplankton blooms represent key events in global carbon cycling. They remove gigatons of atmospheric CO2 and trigger successive blooms of heterotrophic bacterioplankton clades that remineralize most of the fixed carbon. Prior to POMPU we studied such successions during four spring blooms off the North Sea island Helgoland (2009-2012). Using an integrated OMICS approach, we found that recurrently abundant bacterioplankton clades exhibited notably different substrate spectra. This was particularly evident with respect to gene frequencies and expression of carbohydrate-active enzymes (CAZymes) in Bacteroidota, indicating niche partitioning regarding algal polysaccharide degradation. In Bacteroidota the genes that encode the machinery for uptake and degradation of polysaccharides are usually organized in dedicated loci termed ‘polysaccharide utilization loci’ (PULs), and PUL analyses can potentially predict the targeted polysaccharide class.Our role in POMPU thus far was to study marine polysaccharide utilization by comparative genomic analyses of (i) isolates and (ii) of size-fractionated microbial communities. In the first project phase we analyzed (i) PUL repertoires of 53 sequenced North Sea Bacteroidota strains and (ii) a time series of 38 binned and taxonomically classified North Sea metagenomes in order to uncover the PUL repertoires of all abundant bacteria in the 0.2-3 μm (free-living) fraction, including yet uncultivated Bacteroidota. During the second project phase we shifted our focus towards (i) the ecological niches of Gammaproteobacteria during Helgoland spring blooms (ii) comparative genomics of free-living versus particle-associated Bacteroidota (3- 10 μm and >10 μm fractions) during the 2018 spring bloom, and (iii) gene expression analysis of distinct free-living bacterioplankton clades with substantially improved temporal resolution during the 2020 spring bloom using a combination of metagenomics, -transcriptomics, and - proteomics (A1, Z). The latter two approaches generated comprehensive datasets whose analyses will result in integrated ecological interpretations of two very contrasting blooms.During the POMPU extension we want to finalize our analyses of the 2018 and 2020 blooms and complement the 2020 study with data on particle-attached bacteria from the 3- 10 μm size fraction, in particular with respect to obtaining higher quality MAGs (metagenome-assembled genomes) and high temporal resolution metatranscriptome data. These data will also provide a basis for ongoing work packages in subprojects B3, B2 and A1. In addition, we will focus on detailed studies of as yet unexplored polysaccharide-degrading clades, integrate saccharide measurements (A4) with OMICS data and assist geneFISH experiments to visually assign selected PULs to free-living or particle-attached bacteria (B2).