Antibody-mediated isolation and investigation of CPR bacteria

候选的Phyla Radiation(CPR)细菌估计占地球上已编目的总生物多样性的26%。然而，CPR中几乎所有的主要血统都缺乏孤立的代表。应在该项目内开发一种方法，在实验室中专门捕获、共分离和培养CPR细菌及其宿主细菌，以获得对其生理的初步了解。CPR细菌的特点通常是细胞体积小，基因组小，预计是厌氧菌。它们往往缺乏必要的生物合成簇，导致预测它们可能需要与其他细菌、古生菌甚至真核生物共生。然而，到目前为止，几乎所有的发现都来自于基于基因组的观察。为了评估这一单一系统但代谢多样化的群体的生理和生化，有必要开发技术来分离CPR代表并在实验室条件下与其宿主共同培养。为了实现这一点，将从实验室附近的一个地点收集环境样本，那里显示出CPR细菌的高度丰富。来自该部位的细胞将用荧光标记的抗体处理，这些抗体是针对CPR细菌中MAG和SAG衍生的膜蛋白的胞外区而提出的。标记的细胞将通过流式细胞仪进行分选，分析CPR阳性，并在不同的厌氧条件下培养。然后，将对含有生长CPR代表的培养试验进行生理分析。利用荧光和低温电子显微镜对CPR细菌与其共生宿主的共培养进行分析，以了解CPR细菌与其宿主的形态和相互作用。在该项目的一个合作部分，生长的CPR细菌培养上清液将进行第一次新陈代谢分析。纯共培养最终将用于基因组测序，并将构建代谢模型。将对基因组序列进行筛选，寻找新的抗体靶点，以优化抗体促进的CPR细菌捕获。总而言之，项目工作流程分为以下三个部分：A部分：CPR细菌的样本采集、处理、细胞标记和分选B部分：CPR分离株的培养和生理生化分析C部分：单细胞基因组测序、代谢重建和开发用于CPR捕获的新抗体将重复使用新开发的抗体来捕获、分离和表征更多CPR细菌，并评估更多CPR分离株的生理和生化。因此，该项目将产生一种新的工具，使CPR细菌可用于实验室培养，并首次揭示这一广泛但神秘的细菌群体的生理学。

The Candidate Phyla Radiation (CPR) bacteria account for an estimated 26 percent of the total catalogued biodiversity on Earth. Yet, nearly all major lineages within the CPR are lacking isolated representatives. A method shall be developed within this project to specifically capture, co-isolate and cultivate CPR bacteria with their host bacteria in the laboratory to gain first insights into their physiology. CPR bacteria are generally characterized by a small cell size, small genomes and are predicted to be anaerobes. They often lack essential biosynthetic clusters, leading to the prediction that they may require a symbiosis with other bacteria, archaea and even eukaryotes. However, almost all findings stem from genome-based observations so far. To assess the physiology and biochemistry of this monophyletic but metabolically diverse group, it is essential to develop techniques to isolate CPR representatives and co-cultivate them together with their host under laboratory conditions. To achieve this, environmental samples will be collected from a site close to the laboratory, which shows a high abundance in CPR bacteria. The cells from the site will be treated with fluorescently labeled antibodies which were raised against MAG- and SAG-derived extracellular domains of membrane proteins in CPR bacteria. The labeled cells will be sorted via flow cytometry, analyzed for CPR positives and cultivated under different anaerobic conditions. Cultivation assays containing growing CPR representatives will then be subjected to physiological analyses. The co-cultures of a CPR bacterium and its symbiotic host will be analyzed using fluorescence and cryogenic transmission electron microscopy to resolve the morphology and interaction of the CPR bacterium and its host. In a collaborative part of the project, the supernatant of growing CPR bacterial cultures will undergo first metabolomic analyses. The pure co-cultures will finally be used for genome sequencing and metabolic models will be constructed. The genome sequences will be screened for new antibody targets to optimize the antibody-facilitated capture of CPR bacteria. In summary, the project workflow is structured into the following three parts:Part A: Sample collection, processing, cell labeling and sorting of CPR bacteriaPart B: Cultivation and physiological and biochemical analyses of CPR isolatesPart C: Single cell genome sequencing, metabolic reconstruction and development of new antibodies for CPR captureThe workflow will be repeated using the newly developed antibodies to capture, isolate and characterize more CPR bacteria and assess the physiology and biochemistry of more CPR isolates. The project will thus result in a new tool to make CPR bacteria accessible for laboratory cultivations and shed first light onto the physiology of this widespread and yet enigmatic group of bacteria.