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 yet 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 genome size of ∼11 kb and ∼83.3% nucleotide similarity calculated using Virus Intergenomic Distance Calculator (VIRIDIC). Although gene content and synteny were broadly conserved across the isolates, the EPIPs were categorized into distinct groups based on VIRIDIC similarity scores, gene content, and synteny. Notably, EPIPs 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 are distinct from other phage satellite classes. Transmission electron microscopy of EPIPs reveals that they not only feature smaller capsids compared to HerbertWM, but that they feature highly flexible tails of variable length. Additionally, greater EPIP protein similarity to proteins in bacteria as opposed to free phage proteins suggests a functional or evolutionary relationship may exist. The nucleotide similarity, synteny, and unique gene content of EPIPs suggest EPIPs are a distinct class of phage satellites that induce Mycobacteriaceae prophages.