A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc2155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.