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Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

ABSTRACT: There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (?greA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA. Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.

SUBMITTER: Feng S

PROVIDER: S-EPMC7104715 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

Feng Siyuan S Liu Yan Y Liang Wanfei W El-Sayed Ahmed Mohamed Abd El-Gawad MAE Zhao Zihan Z Shen Cong C Roberts Adam P AP Liang Lujie L Liao Liya L Zhong Zhijuan Z Guo Zhaowang Z Yang Yongqiang Y Wen Xin X Chen Hongtao H Tian Guo-Bao GB

Frontiers in microbiology 20200323

There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (ΔgreA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardati ...[more]

PMID: 32265867

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Project description:ObjectivesWolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains.Methodology/principal findingsBiophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with ?2??'? subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with ?2??'? subunits of polymerase.Conclusions/significanceTo the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this ?-proteobacteria and in deciphering the role of Wol GreA in filarial development.

Dataset Information

Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

Publications

Involvement of Transcription Elongation Factor GreA in <i>Mycobacterium</i> Viability, Antibiotic Susceptibility, and Intracellular Fitness.

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