## 3 Mycobacteriumophages--General Characteristics and Overview
These phages exhibited significant nucleotide sequence variability [56; 57]. It is estimated that the genome length of mycobacteriophages ranges from approximately 42 kbp to 150 kbp, with the average length being about 70 kbp. The average GC content of these genomes is 63.7%, with exceptions for phages such as Wildcat (59%) and Rosebush (69%). Mycobacteriumages also vary in the number of tRNA genes they carry. It has been found that about two-thirds of them do not contain these genes, while the remaining one-third (mainly from the _Myoviridae_ family) may encode 30 or more tRNA genes [44]. A comparative analysis of the sequenced genomes of mycobacteriophages revealed a high level of diversity, even greater than that observed in phages infecting _Pseudomonas_ or _Staphylococcus_ species [44]. This diversity may arise from the large number of mycobacterial species susceptible to phage infection, or as suggested, it could be due to differences in laboratory procedures used during phage isolation [44]. As of the latest update from PhagesDB, mycobacteriophages are grouped into more than 30 clusters, labeled from A to AE, based on nucleotide sequence similarity and gene content [58]. Each cluster contains phages with related genomic architecture, and several large clusters--such as cluster A--are further divided into subclusters (e.g., A1 to A9). In addition to these clusters, over 100 singletons have been identified. These are phages whose genomes do not share a sufficient similarity with any other known phages to be assigned to an existing cluster. This classification system continues to evolve as new genomes are sequenced, and it is expected that further diversity, including entirely new clusters and subclusters, will emerge [56]. Therefore, current cluster designations should be regarded as provisional and subject to regular updates. Mycobacteriophage typing has revealed that some phages have a broad host range (e.g., D29), while others infect only a single species of bacteria (e.g., Barnyard, DS6A). This is because phages are capable of recognizing specific receptors on the surface of the bacterial cells they infect [42; 50]. Mycobacteriophages can follow two distinct infection pathways: lytic or lysogenic, with most of them operating in a mild manner (e.g., L5, phages from cluster A), while others lead to the death of the infected cell (e.g., D29). There are also mycobacteriophages, such as Bxz1 and its related phages, whose infection pathway is still unclear [42]. To improve the clinical relevance of mycobacteriophages--particularly in the context of treating _M. tuberculosis_--genetic engineering is increasingly applied to enhance phage safety, efficacy, and precision. A significant proportion of naturally occurring mycobacteria-riophages are temperate, meaning they can integrate into the host bacterial genome via site-specific recombination [59; 60]. Such lysogenic phages pose inherent risks for therapeutic applications due to their potential to mediate horizontal gene transfer, promote bacterial persistence, or elicit unintended genomic modifications in the target host. To address these concerns, temperate phages can be genetically engineered to adopt an obligately lytic lifestyle. This is typically achieved through targeted deletions or disruptions of key regulatory genes, such as integrase and repressor. The integrase enzyme mediates the site-specific recombination between the phage genome and the host chromosome, a critical step for lysogeny. Its removal prevents genomic integration, thus blocking the establishment of lysogeny. Similarly, disruption of the repressor gene eliminates the ability to maintain the lysogenic state, ensuring that the phage proceeds exclusively through the lytic cycle and consistently induces host cell lysis [61; 62]. Further advances in synthetic biology have allowed for the customization of phage genomes to include functional payloads such as CRISPR-Cas systems, antimicrobial peptides, or reporter genes [63; 64].