Construction of EF-G knockdown strain of <i>Mycobacterium smegmatis</i> and drug resistance analysis.

Yuchang DI; Jiacheng Bai; Mingzhe Chi; Weixing Fan; Xuelian Zhang

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Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis.

Author: Yuchang DI ¹ ; Jiacheng BAI ¹ ; Mingzhe CHI ¹ ; Weixing FAN ² ; Xuelian ZHANG ¹
Author Information

1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
2. Laboratory of Zoonosis, China Animal Health and Epidemiology Center, Qingdao 266032, Shangdong, China.
Publication Type:Journal Article
Keywords: EF-G; Mycobacterium smegmatis; clustered regularly interspaced short palindromic repeats interference (CRISPRi); drug resistance; minimal inhibitory concentration (MIC)
MeSH: Antitubercular Agents/pharmacology*; Bacterial Proteins/metabolism*; Drug Resistance; Mycobacterium smegmatis/metabolism*; Peptide Elongation Factor G/pharmacology*
From: Chinese Journal of Biotechnology 2022;38(3):1050-1060
CountryChina
Language:Chinese
Abstract: As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.