The unique N-terminal region of Mycobacterium tuberculosis sigma factor A plays a dominant role in the essential function of this protein

Abstract

SigA (σ^A) is an essential protein and the primary sigma factor in Mycobacterium tuberculosis (Mtb). However, due to the absence of genetic tools, our understanding of the role and regulation of σ^A activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σ^A in Mtb and showed that its depletion results in a severe survival defect in vitro, ex vivo, and in vivo in a murine infection model. Our RNA-seq analysis suggests that σ^A either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes. Surprisingly, we note that despite having ∼64% similarity with σ^A, overexpression of the primary-like σ factor SigB (σ^B) fails to compensate for the absence of σ^A, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σ^B deletion mutant revealed that 433 genes are regulated by σ^B, of which 283 overlap with the σ^A transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σ^A residues between 132-179 that are disordered and missing from all experimentally determined σ^A-RNAP structural models are imperative for σ^A function. Moreover, phosphorylation of σ^A in the intrinsically disordered N-terminal region plays a regulatory role in modulating its activity. Collectively, these observations and analysis provide a rationale for the centrality of σ^A for the survival and pathogenicity of this bacillus.

Keywords: Mycobacteria; Regulation; SigA; SigB; Sigma factor; Transcription; Tuberculosis.