Unique episymbiotic relationship betweenGracilibacteriaandZoogloeain activated sludge flocs in a municipal wastewater treatment plant

Abstract Among the various bacteria present in activated sludge, uncultivated Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) are ubiquitous at the class or phylum level. Patescibacteria have a highly restricted metabolic capacity and are thought to be episymbiotic/endosymbiotic or predatory. However, only a limited number of Patescibacteria and their hosts have been identified. Therefore, many Patescibacteria have not been (co-)cultured and identified by fluorescence in situ hybridization (FISH) or electron microscopy. Little is known about the morphology, metabolic potential, and hosts of Gracilibacteria (formerly GN02 or BD1-5) which belong to Patescibacteria . In our previous study, we confirmed the presence of Gracilibacteria in activated sludge and successfully recovered its high-quality genome. In this study, we designed new probes to visualize members of Gracilibacteria in activated sludge and identified its host using FISH. The FISH observations revealed that Gracilibacteria , which formed loosely associated clusters, were located within dense clusters of Zoogloea , which were dominant in the activated sludge. The metagenome-assembled genomes (MAGs) of Zoogloea possessed genes related to extracellular polymeric substance (EPS) biosynthesis, floc formation, and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The MAGs of Gracilibacteria possessed genes associated with type IV pili, competence protein EC (ComEC), and PHA degradation, which suggests that they have a Zoogloea -dependent lifestyle in activated sludge flocs. These findings clearly indicate a new symbiotic relationship between Gracilibacteria and Zoogloea , and to the best of our knowledge, this is the first study to show this interaction.