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Updated: Jun 20, 2025
Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
Published on: May 29, 2016
Construction of knockout mutants in Mycobacterium intracellulare ATCC13950 strain using a thermosensitive plasmid
Yoshitaka Tateishi1, Akihito Nishiyama1, Yuriko Ozeki1
1Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan.
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.
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