This study describes a novel mechanism of temperate bacteriophage lysogeny called tRNA-dependent lysogeny. The research focuses on bacteriophages that integrate into bacterial chromosomes at sites overlapping host tRNA genes. These phages encode their own tRNA genes of the same isotype as the host tRNA, which is essential for the survival of lysogenic progeny. The phage-encoded tRNA compensates for the loss of host tRNA function during integration, allowing for the establishment of lysogeny. The study shows that phage-encoded tRNAs are crucial for lysogeny, as their absence leads to a clear plaque phenotype and obligate lytic growth. In contrast, phages with intact tRNA genes form turbid plaques and exhibit stable lysogeny. The research also demonstrates that integration-proficient plasmids derived from these phages must carry a cognate phage tRNA gene for efficient integration. These plasmids may be useful tools for mycobacterial genetics. The study identifies tRNA-dependent lysogeny in multiple groups of related viruses, suggesting it may be prevalent in the broader phage community. The findings highlight the importance of tRNA genes in phage-bacterium interactions and provide new insights into the regulation of phage life cycles. The research also reveals that tRNA-dependent lysogeny is a notable departure from the well-studied phage lambda prototype, which does not use tRNA genes or integrate at sites overlapping host tRNA genes. The study underscores the diversity of regulatory systems used by temperate phages and the need for further investigation into the mechanisms of tRNA-dependent lysogeny.This study describes a novel mechanism of temperate bacteriophage lysogeny called tRNA-dependent lysogeny. The research focuses on bacteriophages that integrate into bacterial chromosomes at sites overlapping host tRNA genes. These phages encode their own tRNA genes of the same isotype as the host tRNA, which is essential for the survival of lysogenic progeny. The phage-encoded tRNA compensates for the loss of host tRNA function during integration, allowing for the establishment of lysogeny. The study shows that phage-encoded tRNAs are crucial for lysogeny, as their absence leads to a clear plaque phenotype and obligate lytic growth. In contrast, phages with intact tRNA genes form turbid plaques and exhibit stable lysogeny. The research also demonstrates that integration-proficient plasmids derived from these phages must carry a cognate phage tRNA gene for efficient integration. These plasmids may be useful tools for mycobacterial genetics. The study identifies tRNA-dependent lysogeny in multiple groups of related viruses, suggesting it may be prevalent in the broader phage community. The findings highlight the importance of tRNA genes in phage-bacterium interactions and provide new insights into the regulation of phage life cycles. The research also reveals that tRNA-dependent lysogeny is a notable departure from the well-studied phage lambda prototype, which does not use tRNA genes or integrate at sites overlapping host tRNA genes. The study underscores the diversity of regulatory systems used by temperate phages and the need for further investigation into the mechanisms of tRNA-dependent lysogeny.