2024/07/23 by Yoshitaka Tateishi, Akihito Nishiyama, Yuriko Ozeki +1
Biochemistry, Genetics and Molecular Biology · Medicine · #Mycobacterium research and diagnosis #RNA and protein synthesis mechanisms #Tuberculosis Research and Epidemiology
paper · pdf · doi:10.1111/1348-0421.13167
openalex publication_date 2024/07/23 · openalex created_date 2024/07/24 · openalex updated_date 2026/07/28
Abstract Background Nontuberculous mycobacterial disease has emerged worldwide over the past 20 years. However, there are currently few reports on the established technique for constructing knockout mutants of nontuberculous mycobacteria. Therefore, gene recombination techniques for nontuberculous mycobacteria require further research. Results We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene ( rpsL + ) as a dominant negative selection marker and the hygromycin (Hyg) and lacZ cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in Mycobacterium intracellulare by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor. Conclusions Our findings demonstrate that rpsL + is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature‐sensitive rpsL + method for the construction of knockout mutants will facilitate functional assays to validate the virulence factors of nontuberculous mycobacteria and the pathogenesis of nontuberculous mycobacterial disease.