Functional characterization of the mraW RNA methyltransferase in growth and cell survival of Mycobacterium smegmatis

Abstract

Background: Tuberculosis remains a major global health threat, exacerbated by the increasing prevalence of drugresistant strains. The RNA methyltransferase mraW is predicted to play an important regulator of bacterial gene expression and cellular physiology. However, its function in mycobacteria remains unclear.

Objective: This study aims to characterize the function of the mraW RNA methyltransferase in Mycobacterium smegmatis using a CRISPR interference (CRISPRi)-mediated gene knockdown.

Methods: A mraW-targeting sgRNA was constructed and expressed in M. smegmatis mc²155, and knockdown effect was induced using anhydrotetracycline (ATc). Knockdown efficiency was confirmed by RT-qPCR, which showed approximately 55% reduction in mraW transcript levels.

Results: Phenotypic analysis revealed that mraW knockdown resulted in ATc concentration-dependent growth inhibition, reduced colony formation, and decreased bacterial viability in both solid and liquid culture conditions. Consistent with the inducible nature of the CRISPRi system, stronger growth defects were observed at higher ATc concentrations, suggesting increased repression of mraW, while pLJR962 vector controls remained unaffected, confirming that the observed effects were specifically associated with mraW knockdown. These findings demonstrate that mraW is important for optimal growth and survival in M. smegmatis, supporting a role for RNA methyltransferases in essential mycobacterial physiological processes.

Conclusion: This study further highlights the utility of CRISPRi for functional characterization of essential genes in mycobacteria and suggests that RNA methylation pathways may represent novel potential targets for future anti-tuberculosis strategies.