A distinct class of conjugative megaplasmids is associated with prophages in distantly related bacterial lineages

Closely related prophages have been found in phylogenetically distant bacteria in the human gut, despite limited evidence of productive phage infections across broad host ranges. It remains enigmatic how the wide distribution of prophages could emerge. Here, we identify a previously unrecognized association between a distinct group of conjugative megaplasmids and prophage genomes. We describe two related but diverged groups of conjugative megaplasmids (>300 kilobases) in the human gut microbiome, which we named Hodors. Hodors encode conserved replication, partitioning, and type IV secretion systems, along with a complex surface-associated gene module. A subset of Hodors harbors complete, intact prophage genomes, and closely related prophages (≥95% nucleotide identity) were detected across phylogenetically distant lineages within the bacterial phylum Bacillota, including both Bacilli and Clostridia. We detected signatures consistent with extracellular particle formation and transcriptional activity of Hodor-associated prophages. Our findings suggest that conjugative megaplasmids could contribute to the movement of related prophage genomes between distantly related bacteria, potentially explaining the widespread occurrence of closely related prophages in phylogenetically distant gut bacteria and the apparent uncoupling between lysogenic and infective host ranges. Closely related prophages can be found in phylogenetically distant bacteria, despite limited evidence of phage infections across broad host ranges. Here, Yuan et al. identify a group of conjugative megaplasmids associated with prophages across distantly related gut bacteria, suggesting a potential route for prophage dissemination in the human gut microbiome.