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 <i>greA</i> gene in <i>Mycobacterium</i> spp. The <i>greA</i> mutant strain (Δ<i>greA</i>) was generated in <i>Mycobacterium smegmatis</i>, <i>Mycobacterium tuberculosis</i> (MTB) H37Ra, and <i>M. tuberculosis</i> H37Rv. Deletion of <i>greA</i> results in growth retardation and poor survival in response to adverse stress, besides rendering <i>M. tuberculosis</i> more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting <i>greA</i> results in the differential regulation of 195 genes in <i>M. smegmatis</i> 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 <i>Escherichia coli</i> GreA, <i>M. smegmatis</i> GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of <i>glgX</i>, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of <i>glgX</i> by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of <i>greA</i>. Our data indicates that GreA is an important factor for the survival and resistance establishment in <i>Mycobacterium</i> spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.