Novel crAssphage isolates exhibit conserved gene order and purifying selection of the host specificity protein.

Bacteroidota are the most common bacteria in the human gut and are responsible for degrading complex polysaccharides that would otherwise remain undigested. The abundance of Bacteroides in the gut is shaped by phages such as crAssphages that infect and kill them. While close to 600 genomes have been identified computationally, only four have been successfully cultured. Here, we identify and characterize three novel crAssphage species isolated from wastewater and infecting the bacterial host WH2. We named the novel species, (Bc01), (Bc03), and (Bc11) which span two different families and three genera. These phages may not have co-evolved with their respective bacterial hosts. The phages had a conserved gene arrangement with known crAssphages, but gene similarity within phages belonging to the same taxa was highly variable. Across the three species, only two structural genes encoding a hypothetical protein and a tail spike protein were similar. Evolutionary analysis revealed the tail spike protein is undergoing purifying selection and was predicted to bind to a TonB-dependent transporter on the host cell surface, suggesting a role for host specificity. This study expands the known crAssphage isolates and reveals insights into the crAssphage infection mechanism. The availability of pure cultures of multiple crAssphage infecting the same host provides an opportunity to perform controlled experiments on one of the most dominant members of the human enteric virome.