Abstract：MSMEG_4259 and its homologs are widely present in Mycobacterium, and protein sequence analysis revealed that MSMEG_4259 contains a DEDDh 3'-5' exonuclease domain as well as a GIY-YIG domain of nucleotide excision repair endonucleases UvrC, suggesting its possible involvement in DNA replication or repair. To investigate the physiological function of MSMEG_4259, MSMEG_4259 knockout strain were generated in Mycolicibacterium smegmatis using a homologous recombination method. We measured the growth curves of the wild type and the knockout strains and found no difference, suggesting that MSMEG_4259 is not an essential gene for growth of Mycolicibacterium smegmatis in eutrophic culture conditions. We measured the mutation frequencies of rifamycin-resistant mutation in these strains during the logarithmic growth phase as well as after H2O2 treatment by using a fluctuation test. The results showed that, during the logarithmic growth period, the mutation frequencies in the MSMEG_4259 knockout strains were increased 1.9-fold (P<0.05) compared to that of the wild type. The mutator phenotype of knockout strain was able to be complemented by expression of the wild-type MSMEG_4259. After H2O2 treatment, the mutation frequencies of the wild type and ΔMSMEG_4259 were elevated 3.7- and 2-fold (P<0.001) compared to untreated. These results showed that MSMEG_4259 is involved in the maintenance of genome stability. The results of real-time fluorescence quantitative PCR (qRT-PCR) showed that the transcription of MSMEG_4259 was upregulated 7-fold in wild type upon H2O2 treatment, suggesting that MSMEG_4259 may be involved in the response to DNA oxidative damage. We measured the survival of wild strain and ΔMSMEG_4259 under treatment with ofloxacin, tert-butyl hydrogen peroxide and UV and found that MSMEG_4259 was not involved in the tolerance of Mycolicibacterium smegmatis to these DNA damaging agents. This study reveals the physiological functions of MSMEG_4259 in Mycolicibacterium smegmatis and lays the foundation for further studies on the role of its homolog in the pathogenesis and drug resistance of Mycobacterium tuberculosis.
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