Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen We recently identified two aminoglycoside-acetylating genes [ and ] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. BLASTP analysis identified MAB_2385 as a close homologue of the 3″--phosphotransferase [APH(3″)] from the opportunistic pathogen as a putative streptomycin resistance determinant. Heterologous expression of in increased the streptomycin MIC, while the gene deletion mutant ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in ΔMAB_2385. This demonstrates that encodes a specific and prime innate streptomycin resistance determinant in We further explored the feasibility of applying-based streptomycin counterselection to generate gene deletion mutants in Spontaneous streptomycin-resistant mutants of ΔMAB_2385 were selected, and we demonstrated that the wild-type is dominant over the mutated in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an -based counterselection method in.