The physiological effect of rimI/rimJ silencing by CRISPR interference in Mycobacterium smegmatis mc²155

Area of Science:

• Microbiology
• Biochemistry
• Genetics

Background:

• N-terminal acetylation is a crucial post-translational modification (PTM) in prokaryotes and eukaryotes.
• In bacteria, its regulatory roles in protein stability, gene expression, stress response, and virulence are suggested but mechanistically unclear.
• Mycobacteria utilize RimI/RimJ proteins for N-terminal acetylation.

Purpose of the Study:

• To investigate the physiological impact of N-terminal acetylation on cell survival and stress response in Mycobacterium smegmatis.
• To explore the role of RimI/RimJ proteins in modulating protein acetylation.
• To determine the effect of N-terminal acetylation on antibiotic resistance.

Main Methods:

• CRISPR interference (CRISPRi) was employed to silence rimI/rimJ genes in Mycobacterium smegmatis mc²155.
• Growth curves and biofilm formation assays were conducted for mutant and wild-type strains.
• Proteome and acetylome profiling were performed to analyze global changes.
• Minimum Inhibitory Concentration (MIC) assays were used to assess antibiotic resistance.

Main Results:

• No significant differences in growth curves or biofilm formation were observed between mutant and wild-type strains.
• Total proteome and acetylome profiles revealed significant variations, indicating RimI/RimJ's role in modulating protein acetylation.
• A substantial increase in the minimum inhibitory concentration (MIC) for isoniazid was observed in rimI mutant strains.
• N-terminal acetylation appears to be linked to cellular response against antibiotic stress.

Conclusions:

• CRISPRi is an effective tool for studying PTMs like acetylation in mycobacteria.
• The rimI/rimJ genes are essential for managing cellular responses to antibiotic stress.
• Targeting N-terminal acetylation could be a potential strategy to enhance the efficacy of existing antibiotics.