<i>Mycoplasma bovis</i> <scp>DNA</scp> Glycosylase <scp>MbovP486</scp> Mediates Oxidative <scp>DNA</scp> Damage Repair to Enhance Bacterial Survival

Base excision repair (BER), primarily mediated by DNA glycosylase, is essential for repairing oxidative base lesions generated during antimicrobial immune responses. Despite its significance, the role and mechanisms of DNA glycosylases in Mycoplasma species remain uncharacterized. Here, we report that MbovP486 of Mycoplasma bovis (M. bovis) is a bifunctional DNA glycosylase and apurinic/apyrimidinic (AP) lyase. Functional characterization of the recombinant MbovP486 protein (rMbovP486) demonstrated its ability to bind and cleave DNA containing 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a dose-dependent manner. Site-directed mutagenesis revealed that conserved residues M77, R112, and F114 are critical for DNA binding, while the N-terminal catalytic motif "PELPEV" is essential for DNA cleavage activity. To investigate the physiological relevance of MbovP486, we compared the MbovP486-deficient mutant strain T9.376 with the wild-type strain HB0801 and a complemented strain CT9.376. The T9.376 mutant displayed significantly increased DNA damage and reduced survival following exposure to exogenous hydrogen peroxide (H₂O₂), indicating impaired oxidative DNA repair. Although all three strains similarly induced reactive oxygen species (ROS) production in bovine macrophage (BoMac) cells, the T9.376 mutant exhibited compromised survival under oxidative stress conditions. In conclusion, this study highlights the importance of MbovP486 for M. bovis survival under DNA-damaging conditions during host colonization.