Conditional down-regulation of GreA impacts expression of rRNA and transcription factors, affecting Mycobacterium smegmatis survival

Rajiv Kumar Jha,Ashutosh Kumar Rai,Valakunja Nagaraja,Shamitha Govind,Shubha Udupa,Prakruti R Singh,Phoolwanti Rani

doi:10.1038/s41598-020-62703-7

Abstract

Gre, one of the conserved transcription factors in bacteria, modulates RNA polymerase (RNAP) activity to ensure processivity and fidelity of RNA synthesis. Gre factors regulate transcription by inducing the intrinsic-endonucleolytic activity of RNAP, allowing the enzyme to resume transcription from the paused and arrested sites. While Escherichia coli and a number of eubacteria harbor GreA and GreB, genus mycobacteria has a single Gre (GreA). To address the importance of the GreA in growth, physiology and gene expression of Mycobacterium smegmatis, we have constructed a conditional knock-down strain of GreA. The GreA depleted strain exhibited slow growth, drastic changes in cell surface phenotype, cell death, and increased susceptibility to front-line anti-tubercular drugs. Transcripts and 2D-gel electrophoresis (2D-PAGE) analysis of the GreA conditional knock-down strain showed altered expression of the genes involved in transcription regulation. Among the genes analysed, expression of RNAP subunits (β, β’ and ω), carD, hupB, lsr2, and nusA were affected to a large extent. Severe reduction in the expression of genes of rRNA operon in the knock-down strain reveal a role for GreA in regulating the core components of the translation process.

Highlights

Gre factors or their homologues are conserved in all forms of life, present understanding of their in vivo role in bacteria other than in E. coli is limited
For normalization of qPCR, we used rho mRNA which is abundant in mycobacteria and the protein level did not change during various mycobacterial growth phases and stress (Anirban Mitra, PhD Thesis 2013, Indian Institute of Science, Bangalore)
We describe the effect of reduction in the level of a single transcript cleavage factor GreA in M. smegmatis

Summary

Introduction

Gre factors or their homologues are conserved in all forms of life, present understanding of their in vivo role in bacteria other than in E. coli is limited. Even in E. coli, a complete understanding of the effect of Gre mutants is hindered because of the presence of two Gre (GreA and GreB) and the partial redundancy in function by the other secondary channel binding proteins such as DksA, Rnk and TraR13–15. Presence of a sole representative, lack of some of the other secondary channel binding proteins (DksA, TraR) would likely render the GreA function essential in mycobacteria. Overexpression of antisense RNA of greA in mycobacteria suggested that it could be essential for mycobacterial growth[18]. The conditional knock-down with reduced expression of GreA exhibited growth defect, phenotypic variations, altered protein expression, and cell death indicating the essential role of GreA in maintaining the physiology of M. smegmatis

Methods

Results

Conclusion