Abstract
The generation of conditional mutants has been an effective approach to studying bacteria and validating drug targets, and mutants of Mycobacteria are no exception. However unlike other bacteria, there is still a paucity of available tools for Mycobacteria. We constructed a new plasmid containing tetracycline-repressive expression system (TetRr1.7) and Xer Site-Specific recombinase system to generate label-free controllable expression strains. The plasmid was subsequently used to construct a strain of M. tuberculosis expressing the only copy of d-alanine:d-alanine ligase under the control of the tetracycline-repressive promoter. The results showed that the mutant strain lost the ability of colony formation, became more sensitive to d-cycloserine and the cell wall of the mutant strain was disrupted when anhydrotetracycline was added to the medium. Taken together these observations, confirmed that the expression of d-alanine:d-alanine ligase was tightly controlled by the promoter. In conclusion, the new plasmid is a convenient tool for constructing stable conditional mutant strains in Mycobacteria and can be used for future target identification.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) still remains a heavy threat to human beings
Colony formation efficiency of the mutant Given that the d-alanine:d-alanine ligase (Ddl) gene is essential for Mycobacterium, the ability to form bacterial colonies was investigated in order to verify that the expression of Ddl was controllable
Developing tools for conditional gene regulation is of great importance to gene function research and drug target identification in bacteria
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) still remains a heavy threat to human beings. There were about 10 million patients who developed into tuberculosis and 1 million death every year, many of which were MDR-TB (Kwon 2017). There is an urgent need for new drugs and vaccines to treat the disease. The efforts to exploit new anti-TB drugs seemed hopeless as during the last 50 years only two agents were approved (Lu et al 2014; Blair and Scott 2015). Far only a limited number of targets could be used to develop the drugs, which explains the low success rate.
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