A novel phage satellite class induces prophage excision in Mycolicibacterium aichiense

Phage satellites are mobile genetic elements that parasitize a helper phage to complete their life cycle. While phage satellites are widespread in diverse bacterial hosts, none have been isolated in Mycobacteriaceae . Here, we report the first phage satellites isolated and characterized in Mycobacteriaceae , Extracellular Prophage-Inducing Particles (EPIPs). EPIPs induce a helper phage-HerbertWM, a Mycolicibacterium aichiense prophage-upon infection. Genomic sequencing of thirteen isolates revealed a notably small genome size and high sequence similarity between EPIPs, suggesting they are a distinct class of phage satellite. EPIPs were categorized into groups based on Virus Intergenomic Distance Calculator similarity scores, gene content, and synteny. Gene content is largely conserved across the isolates, though EPIPs notably lack capsid proteins, tail proteins, and holins, suggesting EPIPs hijack the machinery of HerbertWM to replicate, assemble, and lyse the host. Furthermore, the unique gene content of EPIPs compared to other phage satellites, particularly the presence of a tape measure protein and absence of antirepressors, suggests they may exhibit unique mechanisms of assembly and prophage induction. Transmission electron microscopy of EPIPs reveals that they not only feature smaller capsids compared to HerbertWM, but that they feature longer tails of variable length, further suggesting a unique mechanism of tail hijacking that the tape measure protein may facilitate. Additionally, greater EPIP protein similarity to prophage proteins as opposed to free phage proteins suggests a functional or evolutionary relationship may exist. The synteny among EPIPs and the unique gene content suggest EPIPs are a distinct class of phage satellites that induce Mycobacteriaceae prophages.