The annotation of six cluster N Mycobacterium smegmatis phages (Kevin1, Nenae, Parmesanjohn, ShrimpFriedEgg, Smurph, and SpongeBob) reveals regions of genomic diversity, particularly within the central region of the genome. The genome of Kevin1 includes two orphams (genes with no similarity to other phage genes), with one predicted to encode an AAA-ATPase.

Mycobacteriophages Chancellor, Mitti, and Wintermute infect Mycobacterium smegmatis mc2155 and are closely related to phages Cheetobro and Fionnbharth in subcluster K4. Genome sizes range from 57,697 bp to 58,046 bp. Phages are predicted to be temperate and to infect the pathogen Mycobacterium tuberculosis.

Comparative analyses of mycobacteriophage genomes reveal extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. A reverse experiment where an Island3 lysogen was infected with Butters yielded Butters phages and no recombinants, demonstrating directionality to the recombination phenomenon. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for therapeutic use.