Heterologous Substitution of <i>Mycobacterium tuberculosis</i> rRNA in <i>Mycobacterium smegmatis</i> and Its Impact on Antimicrobial Susceptibility

Background : The global incidence of multidrug-resistant and extensively drug-resistant tuberculosis continues to rise. The ribosome serves as a target for multiple antimicrobials, making functional research on it hold great significance. Methods : Using homologous recombination combined with a multiple serine integrase-mediated site-specific recombination system, we replaced the two endogenous rRNA operons in Mycobacterium smegmatis MC 2 155 with a single copy of the single rRNA operon from Mycobacterium tuberculosis H37Rv, constructing the M. smegmatis BRkoA strain. We assessed growth kinetics at 37 °C, cold sensitivity at lower temperatures, transcriptional levels by RT-qPCR, 70S ribosome integrity through cryo-EM, and antimicrobial susceptibility by microdilution assays. Results : The BRkoA strain was successfully constructed. It exhibited markedly slower growth compared to the wild-type strain. Cold-sensitivity assays indicated potential ribosome assembly defects, while transcriptional analysis suggested altered rRNA processing and modification. Cryo-EM analysis further demonstrated the absence of specific ribosomal proteins in the BRkoA 70S ribosome. Moreover, BRkoA displayed reduced susceptibility tendency to several ribosome-targeting antibiotics, including kanamycin, amikacin, paromomycin, gentamicin, and linezolid. Conclusions : Replacement of the two endogenous rrn operons in M. smegmatis with a single copy of the single M. tuberculosis rrn operon using a serine integrase-mediated recombination system caused growth impairment and decreased sensitivity tendency to several ribosome-targeting antimicrobials. These findings suggest that ribosome structural variation contributes to intrinsic drug resistance mechanisms.