Heterologous Substitution of Mycobacterium tuberculosis rRNA in Mycobacterium smegmatis and Its Impact on Antimicrobial Susceptibility

<b>Background</b>: 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. <b>Methods</b>: Using homologous recombination combined with a multiple serine integrase-mediated site-specific recombination system, we replaced the two endogenous rRNA operons in <i>Mycobacterium smegmatis</i> MC² 155 with a single copy of the single rRNA operon from <i>Mycobacterium tuberculosis</i> H37Rv, constructing the <i>M. smegmatis</i> 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. <b>Results</b>: 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. <b>Conclusions</b>: Replacement of the two endogenous <i>rrn</i> operons in <i>M. smegmatis</i> with a single copy of the single <i>M. tuberculosis rrn</i> 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.