Culture- and genome-based characterization of a tripartite interaction between patescibacterial epibionts, methylotrophic proteobacteria, and a jumbo phage in freshwater ecosystems

Patescibacteria form a very diverse and widely distributed phylum of small bacteria inferred to have an episymbiotic lifestyle. However, the prevalence of this lifestyle within the phylum and their host specificity remain poorly known due to the scarcity of cultured representatives. Here, we describe a complex system consisting of a patescibacterium, its gammaproteobacterial hosts, and their respective phages based on enrichment cultures and metagenomic data from two shallow, geographically close, freshwater ecosystems. The patescibacterium, Strigamonas methylophilicida sp. nov., defines a new genus within the family Absconditicoccaceae. It grows as epibiont on cells of methanotrophic species of the gammaproteobacterial family Methylophilaceae. Strigamonas cells grow tightly attached to the host, sometimes forming stacks that connect two host cells. Despite a surprisingly large genome (1.9 Mb) compared to many other Patescibacteria, S. methylophilicida lacks many essential biosynthetic pathways, including the complete biosynthesis of phospholipids, amino acids, and nucleic acids, implying a dependence on the host to obtain these molecules. We also identified and assembled the complete genomes of one patescibacterial phage that might represent a new virus family within the class Caudoviricetes, and two Methylophilaceae phages predicted to have head-tailed and filamentous virions, respectively. The patesciphage uses a modified genetic code similar to that of its host and encodes four tRNA genes, including the suppressor tRNA gene for the UGA stop codon, which is reassigned to glycine in many Patescibacteria. Our results confirm a prevalent episymbiotic lifestyle in Absconditicoccaceae and further suggest a clade-specific adaptation of this patescibacterial family for gammaproteobacterial hosts. IMPORTANCEPatescibacteria are ultra-small bacteria with reduced genomes that rely on symbiotic interactions with other prokaryotes, yet their host specificity and associated viral parasites remain poorly characterized due to limited cultured representatives. Here, we describe Strigamonas methylophilicida, a new patescibacterial species of the family Absconditicoccaceae that grows as an epibiont on various methylotrophic Gammaproteobacteria. This expands the host range for this family beyond previously documented photosynthetic partners. Using enrichment cultures and metagenomics, we retrieved complete genomes of novel phages infecting S. methylophilicida and its hosts, including one phage that uses a modified genetic code matching that of the patescibacterium. These findings reveal a previously unrecognized patescibacteria- methylotroph-phage tripartite interaction in freshwater environments, highlight the adaptations of patescibacterial phages, and shed light on the complex ecology and evolution of host-parasite-phage dynamics in understudied bacterial lineages.