Unique episymbiotic relationship between Candidatus Patescibacteria and Zoogloea in activated sludge flocs at a municipal wastewater treatment plant.

Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class-level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1-5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high-quality metagenome-assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01-associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10-like cells were located within dense clusters of Zoogloea, and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea-dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea.