Mycobacterium tuberculosis m 4 C DNA methyltransferase Rv3204 promotes mycobacteria survival under oxidative stress

Abstract Reactive oxygen species (ROS) inflict cellular damage yet are pivotal mediators of signaling pathways. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), persists as a major global health threat, partly due to its capacity to neutralize host-derived ROS toxicity. While DNA methylation deficiency is known to attenuate Mtb virulence, the specific role of DNA methyltransferases in mycobacterial survival under oxidative stress remains poorly defined. Here, we demonstrate that the mycobacterial protein Rv3204 functions as an N 4 -methylcytosine (m 4 C) DNA methyltransferase. Deletion of the Rv3204 homolog ( Ms_1939 ) in Mycobacterium smegmatis significantly impaired bacterial survival upon rifampicin exposure. This phenotype was associated with heightened intracellular ROS accumulation and a failure to upregulate transcription of ROS detoxification genes. Furthermore, the mutant exhibited downregulated expression of DNA repair genes and increased susceptibility to fluoroquinolone antibiotics (norfloxacin, ofloxacin). Crucially, the Ms_1939 deletion strain displayed elevated levels of DNA damage. To our knowledge, this is the first study establishing a direct link between an m 4 C DNA methyltransferase and ROS homeostasis in mycobacteria. Our findings identify Rv3204 as a potential novel therapeutic target for modulating ROS sensitivity in M. tuberculosis . Importance Mycobacterium tuberculosis , the causative agent of tuberculosis, remains a major global health threat due in part to its ability to withstand host-derived oxidative stress. This study identifies Rv3204 as a novel N4-methylcytosine DNA methyltransferase essential for mycobacterial survival under oxidative stress. We demonstrate that deletion of Rv3204 homolog in M. smegmatis leads to elevated intracellular ROS, impaired transcriptional activation of antioxidant and DNA repair genes, increased DNA damage, and heightened susceptibility to fluoroquinolone antibiotics. Our findings establish a critical link between epigenetic regulation via m 4 C methylation and ROS homeostasis in mycobacteria—a relationship previously unexplored. This work not only advances our understanding of bacterial epigenetic mechanisms in stress adaptation but also positions Rv3204 as a promising target for novel anti-tuberculosis strategies aimed at disrupting redox balance and enhancing antibiotic efficacy. DATA summary Raw data files have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE139646 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139646 ).