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| Construction of trans-translational reporting system in Mycobacterium smegmatis |
| HU Yawen, BAI Jiacheng, GE Wenxue, YAO Mengyi, ZHANG Xuelian |
| State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China |
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Abstract During the translation process, the translation would be terminated prematurely when abnormal mRNA (such as the deletion of the stop codon) is translated, resulting in the ribosome stalling and causing the bacteria to initiate ribosome rescue pathway. The trans-translation system mediated by tmRNA-SmpB is the main ribosome rescue pathway in Mycobacterium tuberculosis (M. tuberculosis), which has a significant impact on its growth and physiology. In order to explore the initiation and other functional characteristics of the trans-translation pathway in mycobacteria, Mycobacterium smegmatis (M. smegmatis) was selected as the experimental strain and a reporting system that reflects trans-translational expression in cells by adding Escherichia coli (E. coli) terminator at the 3′ end of the reporter genes was constructed. mCherry and egfp were chosen as reporter genes respectively. The results showed that the immature mCherry protein expressed in the experimental strain was quickly hydrolyzed compared with the normal mCherry expressed in the control strain, and the color of the former strain was significantly lighter than that of the latter strain. In addition, the quantitative detection data of enhanced green fluorescent protein (EGFP) also showed that the error EGFP level was significantly lower. These results suggested that both trans-translational reporting systems were successfully constructed. Dynamic expression data of the reporter genes showed that M. smegmatis could initiate the trans-translation pathway and completely hydrolyze the immature error proteins at 40 h to 45 h. This study will help us carry out functional researches on the trans-translation pathway of mycobacteria and screen for new anti-tuberculosis drugs.
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Received: 13 November 2018
Published: 25 February 2018
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Corresponding Authors:
ZHANG Xuelian
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2019, Vol. 14 
