Host interactions of novel Crassvirales species belonging to multiple families infecting bacterial host, Bacteroides cellulosilyticus WH2

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the <i>Crassvirales</i> order. Despite identifying over 600 <i>Crassvirales</i> genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more <i>Crassvirales</i> genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various <i>Bacteroides</i> hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel <i>Crassvirales</i> species infecting <i>Bacteroides cellulosilyticus</i> WH2. These species, <i>Kehishuvirus</i> sp. 'tikkala' strain Bc01, <i>Kolpuevirus</i> sp. 'frurule' strain Bc03, and 'Rudgehvirus jaberico' strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured <i>Crassvirales</i> species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three <i>Crassvirales</i> species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome.