An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

A novel synergistic enzyme-antibiotic therapy with immobilization of mycobacteriophage Lysin B enzyme onto Rif@UiO-66 nanocomposite for enhanced inhaled anti-TB therapy; Nanoenzybiotics approach.

The emergence of antibiotic-resistant and phage-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) necessitates improving new therapeutic plans. The objective of the current work was to ensure the effectiveness of rifampicin and the mycobacteriophage LysB D29 (LysB)enzyme in the treatment of multi-drug resistant tuberculosis (MDR-TB) infection, where new and safe metal-organic framework (MOF) nanoparticles were used in combination. UiO-66 nanoparticles were synthesized under mild conditions in which the antimycobacterial agent (rifampicin) was loaded (Rif@UiO-66) and LysB D29 enzyme immobilized onto Rif@UiO-66, which were further characterized. Subsequently, the antibacterial activity of different ratios of Rif@UiO-66 and LysB/Rif@uio-66 against the nonpathogenic tuberculosis model Mycobacterium smegmatis (M. smegmatis) was evaluated by minimum inhibitory concentration (MIC) tests. Impressively, the MIC of LysB/Rif@uio-66 was 16-fold lower than that of pure rifampicin. In vitro and in vivo toxicity studies proved that LysB/Rif@UiO-66 is a highly biocompatible therapy for pulmonary infection. A biodistribution assay showed that LysB/Rif@UiO-66 showed a 5.31-fold higher drug concentration in the lungs than free rifampicin. A synergistic interaction between UiO-66, rifampicin and the mycobacteriophage lysB D29 enzyme was shown in the computational method (docking). Therefore, all results indicated that the LysB/Rif@UiO-66 nanocomposite exhibited promising innovative enzyme-antibiotic therapy for tuberculosis treatment.

Alginate-Encapsulated Mycobacteriophage: A Potential Approach for the Management of Intestinal Mycobacterial Disease.

Encapsulated medication is a common method of administering therapeutic treatments. As researchers explore alternative therapies, it is likely that encapsulation will remain a feature of these novel treatments, particularly when routes of delivery are considered. For instance, alginate-encapsulation is often favoured where gastric digestion poses an obstacle. When exposed to cations (namely Ca2+), alginate readily forms gels that are resilient to acidic conditions and readily dissociate in response to mid-range pH. This action can be extremely valuable for the encapsulation of phages. The efficient delivery of phages to the intestine is important when considering mycobacteriophage (MP) therapy (or MP prophylaxis) for disseminated mycobacterial infections and chronic gastroenteritis conditions. This study presents the design and in vitro validation of an alginate-encapsulated MP capable of releasing phages in a pH-dependent manner. Ultimately, it is shown that encapsulated phages pretreated with simulated gastric fluid (SGF) are capable of releasing viable phages into simulated intestinal fluid (SIF) and thereby reducing the mycobacterial numbers in spiked SIF by 90%. These findings suggest that alginate encapsulation may be a viable option for therapeutic and prophylactic approaches to the management of intestinal mycobacterial disease, such as Johne's disease.

A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy.

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

IMPORTANCE: Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

Mycobacteriophage D29 Lysin B exhibits promising anti-mycobacterial activity against drug-resistant Mycobacterium tuberculosis.

IMPORTANCE: To combat the rapidly emerging drug-resistant M. tuberculosis, it is now essential to look for alternative therapeutics. Mycobacteriophages can be considered as efficient therapeutics due to their natural ability to infect and kill mycobacteria including M. tuberculosis. Here, we have exploited the mycolyl-arabinogalactan esterase property of LysB encoded from mycobacteriophage D29. This study is novel in terms of targeting a multi-drug-resistant pathogenic strain of M. tuberculosis with LysB and also examining the combination of anti-TB drugs and LysB. All the experiments include external administration of LysB. Therefore, the remarkable lytic activity of LysB overcomes the difficulty to enter the complex cell envelope of mycobacteria. Targeting the intracellularly located M. tuberculosis by LysB and non-toxicity to macrophages take the process of the development of LysB as a drug one step ahead, and also, the interaction studies with rifampicin and isoniazid will help to form a new treatment regimen against tuberculosis.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

Characterization of novel recombinant mycobacteriophages derived from homologous recombination between two temperate phages.

Comparative analyses of mycobacteriophage genomes reveals 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. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.

Therapeutically useful mycobacteriophages BPs and Muddy require trehalose polyphleates.

Mycobacteriophages show promise as therapeutic agents for non-tuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces or mechanisms of phage resistance. We show here that trehalose polyphleates (TPPs)-high-molecular-weight, surface-exposed glycolipids found in some mycobacterial species-are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy. TPP loss leads to defects in adsorption and infection and confers resistance. Transposon mutagenesis shows that TPP disruption is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss by mutation, and some M. abscessus clinical isolates are naturally phage-insensitive due to TPP synthesis gene mutations. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

Isolation and characterization of a novel mycobacteriophage Kashi-VT1 infecting Mycobacterium species.

Mycobacteriophages are viruses that infect members of genus Mycobacterium. Because of the rise in antibiotic resistance in mycobacterial diseases such as tuberculosis, mycobacteriophages have received renewed attention as alternative therapeutic agents. Mycobacteriophages are highly diverse, and, on the basis of their genome sequences, they are grouped into 30 clusters and 10 singletons. In this article, we have described the isolation and characterization of a novel mycobacteriophage Kashi-VT1 (KVT1) infecting Mycobacterium >smegmatis mc2 155 (M. smegmatis) and Mycobacterium fortuitum isolated from Varanasi, India. KVT1 is a cluster K1 temperate phage that belongs to Siphoviridae family as visualized in transmission electron microscopy. The phage genome is 61,010 base pairs with 66.5% Guanine/Cytosine (GC) content, encoding 101 putative open reading frames. The KVT1 genome encodes an immunity repressor, a tyrosine integrase, and an excise protein, which are the characteristics of temperate phages. It also contains genes encoding holin, lysin A, and lysin B involved in host cell lysis. The one-step growth curve demonstrated that KVT1 has a latency time of 90 min and an average burst size of 101 phage particles per infected cell. It can withstand a temperature of up to 45°C and has a maximum viability between pH 8 and 9. Some mycobacteriophages from cluster K are known to infect the pathogenic Mycobacterium tuberculosis (M. tuberculosis); hence, KVT1 holds potential for the phage therapy against tuberculosis, and it can also be engineered to convert into an exclusively lytic phage.

Virion glycosylation influences mycobacteriophage immune recognition.

Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus infections.

Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

Exploring Host-Binding Machineries of Mycobacteriophages with AlphaFold2.

Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies.

A novel nucleic acid-binding protein, Gp49, from mycobacteriophage with mycobactericidal activity has the potential to be a therapeutic agent.

The mycobacteriophages encode unique proteins that are potent to be therapeutic agents. We screened several clones with mycobactericidal properties from a genomic library of mycobacteriophages. Here we report the properties of one such clone coding the gene product, Gp49, of the phage Che12. Gp49 is a 16 kD dimeric protein having an HTH motif at its C-terminal and is highly conserved among mycobacteriophages and likely to be part of phage DNA replication machinery. Alphafold predicts it to be an α-helical protein. However, its CD spectrum showed it to be predominantly ß-sheeted. It is a high-affinity heparin-binding protein having similarities with the macrophage protein Azurocidin. Its ß-sheeted apo-structure gets transformed into α-helix upon binding to heparin. It binds to linear dsDNA as well as ssDNA and RNA cooperatively in a sequence non-specific manner. This DNA binding property enables it to inhibit both in vitro and in vivo transcription. The c-terminal HTH motif is responsible for binding to both heparin and nucleic acids. Its in vivo localization on DNA could cause displacements of many DNA-binding proteins from the bacterial chromosome. We surmised that the bactericidal activity of Gp49 arises from its non-specific DNA binding leading to the inhibition of many host-DNA-dependent processes. Its heparin-binding ability could have therapeutic/diagnostic usages in bacterial sepsis treatment.

Mycobacteriophage Derived Lipoarabinomannan Binding Protein for Recognizing Non-Tuberculosis Mycobacteria.

Non-tuberculosis mycobacteria (NTM) is one family of pathogens usually leading to nosocomial infections. Exploration of high-performance biological recognition agent plays a pivotal role for the development of point-of-care testing device and kit for detecting NTM. Mycobacterium smegmatis (M. smegmatis) is a NTM which has been frequently applied as an alternative model for highly pathogenic mycobacteria. Herein, a recombinant tail protein derived from mycobacteriophage SWU1 infecting M. smegmatis was expressed with Escherichia coli system and noted as GP89. It shows a fist-like structure according to the results of homology modeling and ab initio modeling. It is confirmed as a lipoarabinomannan (LAM) binding protein, which can recognize studied NTM genus since abundant LAM constructed with d-mannan and d-arabinan is distributed over the mycobacterial surface. Meanwhile an enhanced green fluorescent protein (eGFP)-fused GP89 protein was acquired with a fusion expression technique. Then GP89 and eGFP-fused GP89 were applied to establish a sensitive and rapid method for fluorescent detection of M. smegmatis with a broad linear range of 1.0 × 102 to 1.0 × 106 CFU mL-1 and a low detection limit of 69 CFU mL-1. Rapid and reliable testing of antimicrobial susceptibility was achieved by the GP89-based fluorescent method. The present work provides a promising recognition agent for studied NTM and opens an avenue for clinical diagnosis of NTM-induced infections.

Characterization and genome analysis of G1 sub-cluster mycobacteriophage Lang.

Phage therapy is revitalized as an alternative to antibiotics therapy against antimicrobials resistant pathogens. Mycobacteriophages are genetically diverse viruses that can specifically infect Mycobacterium genus including Mycobacterium tuberculosis and Mycobacterium smegmatis. Here, we isolated and annotated the genome of a mycobacteriophage Lang, a temperate mycobacteriophage isolated from the soil of Hohhot, Inner Mongolia, China, by using Mycolicibacterium smegmatis mc2 155 as the host. It belongs to the Siphoviridae family of Caudovirales as determined by transmission electron microscopy. The morphological characteristics and certain biological properties of the phage were considered in detail. Phage Lang genomes is 41,487 bp in length with 66.85% GC content and encodes 60 putative open reading frames and belongs to the G1 sub-cluster. Genome annotation indicated that genes for structure proteins, assembly proteins, replications/transcription and lysis of the host are present in function clucters. The genome sequence of phage Lang is more than 95% similar to that of mycobacteriophage Grizzly and Sweets, differing in substitutions, insertions and deletions in Lang. One-step growth curve revealed that Lang has a latent period of 30 min and a outbreak period of 90 min. The short latent period and rapid outbreak mark the unique properties of phage Lang, which can be another potential source for combating M. tuberculosis.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

Effective Isolation and Characterization of Mycobacteriophages with the Ability to Lyse Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Currently there is no cure for JD and test-based culling has proved ineffective at preventing the spread. To isolate new mycobacteriophages (mbps) that can potentially be used to control JD transmission and infection on dairy farms, we optimized an isolation protocol by fecal spiking and the testing of different isolation solution compositions. Using this protocol, we successfully enhanced the yield of mbps from spiked fecal samples, elevating it from less than 1% to 59%. With this method, we isolated 14 mbps from 475 environmental samples collected from MAP-positive dairy farms, after in-sample enrichment with MAP and the fast-growing M. smegmatis. The sample sources included soil, manure pits, lactation barns, feces, milk, and drain water. After fingerprinting these mbps by restriction enzyme profiling, we concluded that 12 were distinct and novel. Further characterization of their host range revealed that eight were capable of lysing multiple MAP strains. We also studied the cross-resistance, lysogeny, the effect of pH and their antimycobacterial properties in milk replacer. Each novel mbp showed limited cross-resistance and prophage immunity and showed no reduction in the titer in a range of pHs after 4 h. The novel phages were also able to reduce the mycobacterial counts to zero after 8 h in milk replacer. In conclusion, these novel mbps could be considered to be used in the control strategies of JD on farms.

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages.

Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages-and thus one of the selective pressures acting on complex microbial systems in nature-remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from 6 subclusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of subcluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of subclusters P2-P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles-a temperate characteristic that impedes their usage in antibacterial applications.