Transcriptomics reveals the role of NHEJ in mycobacterial antiphage defense

In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break (DSB) repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance towards high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium-phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution. Overall design: M. smegmatis and its ?ku?ligD were cultured in 7H9 medium to an OD600 of 2.0. Bacterial cells were collected by centrifugation and resuspended in fresh 7H9 medium to an OD600 of 1.0. The cultures were then infected with SWU1 phage at an MOI of 10, followed by a 30 minutes adsorption period at 37?. Subsequent incubation was carried out at 37°C with shaking at 200 rpm for 30 and 180 minutes. At each time point, bacteria were harvested by centrifugation, and total RNA was extracted for RNA-seq analysis.