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

子项目B1主要研究多糖降解细菌的比较基因组学。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，并引发了异养细菌（浮游生物枝）的连续繁殖，这些细菌使大多数固定碳再矿化。在POMPU之前，我们在北海Helgoland岛（2009-2012）的四个春季花期间研究了这种演替。利用集成的组学方法，我们发现反复丰富的浮游细菌分支表现出明显不同的底物光谱。这在拟杆菌门中碳水化合物活性酶（CAZymes）的基因频率和表达方面尤为明显，表明了藻类多糖降解的生态位分配。在拟杆菌群中，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”（PULs）的专用位点上，PULs分析可以潜在地预测目标多糖类别。到目前为止，我们在POMPU中的作用是通过比较基因组分析(i)分离物和（ii）大小分离微生物群落来研究海洋多糖的利用。在第一个项目阶段，我们分析了(i) 53个已测序的北海拟杆菌属菌株的PUL谱，（ii） 38个分组和分类的北海元基因组的时间序列，以揭示0.2-3 μm（自由生活）馏分中所有丰富细菌的PUL谱，包括尚未培养的拟杆菌属。在第二个项目阶段，我们将重点转移到(i) Helgoland春季水华期间Gammaproteobacteria的生态位；（ii） 2018年春季水华期间自由生活与颗粒相关的拟杆菌门（3- 10 μm和bbb10 μm分数）的比较基因组学；（iii）在2020年春季水华期间，使用宏基因组学、转录组学、生物信息学和生物信息学的组合，对不同的自由生活浮游细菌分支进行基因表达分析，大大提高了时间分辨率。和-蛋白质组学（A1， Z）。后两种方法产生了全面的数据集，其分析将导致对两种截然不同的华花的综合生态解释。在POMPU扩展期间，我们希望完成对2018年和2020年菌华的分析，并利用3- 10 μm大小的颗粒附着细菌的数据来补充2020年的研究，特别是关于获得更高质量的MAGs（宏基因组组装基因组）和高时间分辨率的宏转录组数据。这些数据还将为子项目B3、B2和A1中正在进行的工作包提供基础。此外，我们将专注于尚未探索的多糖降解分支的详细研究，将糖测量（A4）与组学数据相结合，并协助geneFISH实验以视觉方式将选定的PULs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).