25A diverse set of prophage-mediated mechanisms protecting bacterial hosts from 26 infection has been recently uncovered within Cluster N mycobacteriophages. In that 27 context, we unveil a novel defense mechanism in Cluster N prophage Butters. By using 28 bioinformatics analyses, phage plating efficiency experiments, microscopy, and 29 immunoprecipitation assays, we show that Butters genes located in the central region of 30 the genome play a key role in the defense against heterotypic viral attack. Our study 31 suggests that a two component system articulated by interactions between protein 32 products of genes 30 and 31 confers defense against heterotypic phage infection by 33 PurpleHaze or Alma, but is insufficient to confer defense against attack by the 34 heterotypic phage Island3. Therefore, based on heterotypic phage plating efficiencies 35 on the Butters lysogen, additional prophage genes required for defense are implicated. 36 37 IMPORTANCE 38 Many sequenced bacterial genomes including pathogenic bacteria contain prophages. 39 Some prophages encode defense systems that protect their bacterial host against 40 heterotypic viral attack. Understanding the mechanisms undergirding these defense 41 systems will be critical to development of phage therapy that circumvents these 42 defenses. Additionally, such knowledge will help engineer phage-resistant bacteria of 43 industrial importance.44 45 47they are useful in diagnostics of mycobacterial infections (1), the most notable of which 48 is tuberculosis (TB), and additionally can serve as genetic tools for mycobacteria (2-5). 49Most recently, engineered mycobacteriophages have been used in therapeutic 50 3 applications to combat infections from antibiotic-resistant strains of Mycobacterium 51 abscessus (6). To date over 11,000 mycobacteriophages have been isolated, over 52 1,800 sequenced, and over 1,600 are available in GenBank (7, 8). Mycobacteriophages 53 are a small subset of the estimated 10 31 bacteriophages existing in the biosphere (9). 54 Mycobacteriophages display high levels of genetic diversity and have been divided into 55 29 genomically similar clusters (A-AC) and a group of singletons with no close relatives 56 (10, 7). Although an increase in isolation and genomic characterization of 57 mycobacteriophages has occurred recently, the void in knowledge about gene 58 expression and function of mycobacteriophage gene products remains. 59 Prophages make up a majority of the known bacteriophage population (11). The 60 relationship between prophages and bacterial strains has shown numerous benefits to 61 both the hosts and phages. Prophages confer many advantages to the host upon 62 integration such as enhanced fitness, reduction of mutation rates, selective advantages, 63 and defense against additional viral attack (12). The bacterial host in turn provides the 64 phages with protection from harsh environments (12). In this context, numerous 65 mechanisms of defense have been recently discovered for Pseudomonas, 66 Mycobacterium, and Gordonia prophages...

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.

Many sequenced bacterial genomes, including those of pathogenic bacteria, contain prophages. Some prophages encode defense systems that protect their bacterial host against heterotypic viral attack. Understanding the mechanisms undergirding these defense systems is crucial to appreciate the scope of bacterial immunity against viral infections and will be critical for better implementation of phage therapy that would require evasion of these defenses. Furthermore, such knowledge of prophage-encoded defense mechanisms may be useful for developing novel genetic tools for engineering phage-resistant bacteria of industrial importance.

During lysogeny temperate phages establish a truce with the bacterial host. In this state, the phage genome (prophage) is maintained within the host environment. Consequently, many prophages have evolved systems to protect the host from heterotypic viral attack. This phenomenon of prophages mediating defense of their host against competitor phages is widespread among temperate mycobacteriophages. We previously showed that theMycobacteriumphage Butters prophage encodes a two-component system (gp30/31) that inhibits infection from a subset of mycobacteriophages that include PurpleHaze, but not Island3. Here we show that Butters gp57r is both necessary and sufficient to inhibit infection by Island3 and other phages. Gp57r acts post-DNA injection and its antagonism results in the impairment of Island3 DNA amplification. Gp57r inhibition of Island3 is absolute with no defense escape mutants. However, mutations mapping to minor tail proteins allow PurpleHaze to overcome gp57r defense. Gp57r has a HEPN domain which is present in many proteins involved in inter-genomic conflicts, suggesting that gp57r may inhibit heterotypic phage infections via its HEPN domain. We also show that Butters gp57r has orthologues in clinical isolates ofMycobacterium abscessussp. including the phage therapy candidate strain GD91 which was found to be resistant to the panel of phages tested. It is conceivable that this GD91 orthologue of gp57r may mediate resistance to the subset of phages tested. Challenges of this nature underscore the importance of elucidating mechanisms of antiphage systems and mutations that allow for escape from inhibition.

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.