Hypermutability of

Ribonucleotide reductase (RNR) catalyzes the synthesis of four deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication. Although dNTP imbalances reduce replication fidelity and elevate mutation rates, the impact of RNR dysfunction on Mycobacterium tuberculosis (Mtb) physiology and drug resistance remains unknown. Here, we constructed inducible knockdown strains for the RNR R1 subunit NrdE in Mtb and Mycolicibacterium smegmatis (Msm). NrdE knockdown significantly impaired growth and metabolic imbalances, indirectly disrupting oxidative homeostasis and mycolic acid synthesis, while increasing levels of intracellular ROS accumulation and enhancing cell wall permeability. Additionally, we developed genomic mutant strains, Msm-Y252A and Msm-Q255A, featuring targeted point mutations in the substrate-specific site (S-site) of the RNR loop domain, which determines NDP reduction specificity. The Msm-Y252A displayed a 1.9-fold decrease in dATP and increases in dGTP (1.6-fold), dTTP (9.0-fold), and dCTP (1.3-fold). In contrast, Msm-Q255A exhibited elevated intracellular levels of dGTP (1.6-fold), dTTP (6.1-fold), and dATP (1.5-fold), while dCTP levels remained unchanged. Neither the NrdE knockdown strain nor the S-site mutants exhibited direct resistance development; however, they both showed genomic instability, enhancing the emergence of rifampicin-resistant mutants, even with a 70-fold and a 25-fold increase in mutation frequency for Msm-Y252A and Msm-Q255A, respectively. This study demonstrates that NrdE is integral to Mycobacterium survival and genomic stability and that its RNR dysfunction creates a mutagenic environment under selective pressure, indirectly contributes to the development of drug resistance, positioning NrdE as an effective target for therapeutic strategies and a valuable molecular marker for early detection of drug-resistant Mtb.