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 (ΔgreA) 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.
Highlights
Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has influenced human populations since ancient times
To explore the function of greA in mycobacteria, we deleted the greA (MSMEG_5263) gene by allelic exchange facilitated via recombination in M. smegmatis mc2155 which is widely used as a model to study non-pathogenic aspects of mycobacterial physiology
We have detected colonies of small sizes in the greA mutant strain compared with the parental strain (Figure 1A), it was observed that greA strain grows slower than the wildtype M. smegmatis and the phenotypes that were complemented by expression of greA from M. smegmatis on the chromosome (Figure 1B)
Summary
Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has influenced human populations since ancient times. In 2018, M. tuberculosis caused seven million TB cases and was responsible for 1.5 million deaths worldwide (Harding, 2019). The administration of anti-TB drugs has led to the emergence of drug-resistant strains of MTB (Kumar et al, 2017) with approximately 5% of infections being caused by multidrug-resistant (MDR) strains globally (Wright et al, 2009). China has a high prevalence of drug-resistant TB and is the region with second largest number of MDR cases worldwide (Yang et al, 2017). Several transcription factors can bind to RNAP, modifying its properties by affecting transcription processivity and fidelity through modulating pausing, arrest, and termination (Tehranchi et al, 2010; Li et al, 2015). The transcript cleavage factor, GreA, interacts with the RNAP secondary channel and stimulates the intrinsic transcript cleavage activity of RNAP for the removal of the aberrant RNA 3 ends. Polymerization activity can be restarted from the end of a cleaved RNA allowing transcription to resume (Komissarova and Kashlev, 1997)
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