Antibody-mediated isolation and investigation of CPR bacteria

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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.