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

SigA (σA) 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 σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA 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 σA either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes. Surprisingly, we note that despite having ∼64% similarity with σA, overexpression of the primary-like σ factor SigB (σB) fails to compensate for the absence of σA, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σB deletion mutant revealed that 433 genes are regulated by σB, of which 283 overlap with the σA transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σA residues between 132-179 that are disordered and missing from all experimentally determined σA-RNAP structural models are imperative for σA function. Moreover, phosphorylation of σA 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 σA for the survival and pathogenicity of this bacillus. SigA (σA) 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 σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA 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 σA either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes. Surprisingly, we note that despite having ∼64% similarity with σA, overexpression of the primary-like σ factor SigB (σB) fails to compensate for the absence of σA, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σB deletion mutant revealed that 433 genes are regulated by σB, of which 283 overlap with the σA transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σA residues between 132-179 that are disordered and missing from all experimentally determined σA-RNAP structural models are imperative for σA function. Moreover, phosphorylation of σA 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 σA for the survival and pathogenicity of this bacillus. Mycobacterium tuberculosis (Mtb) enters the body through nasal airways and deposits in the lower lungs, wherein the primarily alveolar macrophages engulf the bacteria (1Cambier C.J. Falkow S. Ramakrishnan L. Host evasion and exploitation schemes of Mycobacterium tuberculosis.Cell. 2014; 159: 1497-1509Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). In response to the host's dynamic microenvironment, Mtb remodels its gene expression to facilitate its survival within the host. Gaining insights into how Mtb modulates its transcriptional machinery to survive under hostile host conditions is imperative for tackling this deadly pathogen. Gene expression is primarily regulated at the transcription initiation step in bacteria. Transcription initiation involves many diverse molecular interactions that allow the apo RNA polymerase (RNAP) to recognize the promoter and facilitate DNA unwinding around the transcription start-point. Contrary to eukaryotes, wherein three RNAP are present, bacteria encode only one RNAP consisting of 2 subunits of α, one subunit each of β, β′, and a ω subunit (2Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K. Factor stimulating transcription by RNA polymerase.Nature. 1969; 221: 43-46Crossref PubMed Scopus (649) Google Scholar). Even though core RNAP is sufficient for transcriptional elongation, it cannot initiate transcription without a σ factor (3Hansen U.M. McClure W.R. Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. I. Characterization of core enzyme open complexes.J. Biol. Chem. 1980; 255: 9556-9563Abstract Full Text PDF PubMed Google Scholar). σ factors play a crucial role in promoter recognition and initiating the melting process of promoter regions (4Saecker R.M. Record Jr., M.T. Dehaseth P.L. Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis.J. Mol. Biol. 2011; 412: 754-771Crossref PubMed Scopus (235) Google Scholar, 5Paget M.S. Helmann J.D. The sigma70 family of sigma factors.Genome Biol. 2003; 4: 203Crossref PubMed Scopus (371) Google Scholar, 6Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex.Science. 2002; 296: 1285-1290Crossref PubMed Scopus (543) Google Scholar, 7Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. et al.The functional and regulatory roles of sigma factors in transcription.Cold Spring Harbor Symp. Quant. 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However, it the survival of Mtb in macrophages or (1Cambier C.J. Falkow S. Ramakrishnan L. Host evasion and exploitation schemes of Mycobacterium tuberculosis.Cell. 2014; 159: 1497-1509Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, S. I. expression of sigma factor genes in Mycobacterium PubMed Scopus Google Scholar, Transcription of sigma and in Mycobacterium PubMed Google Scholar, M. M. et al.The Mycobacterium tuberculosis sigma factor is for response to and in vitro, it is for in vivo PubMed Scopus Google Scholar). that are by σA and σB SigA and SigB essential genes PubMed Scopus Google Scholar). σA and σB are J. J. S. et and and essential Mycobacterium tuberculosis and PubMed Scopus Google Scholar, J. 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However, the N-terminal in σA is at is in interactions of σA with the promoter Record Jr., M.T. C.A. regions of transcription initiation factor sigma of to promoter Full Text PDF PubMed Scopus Google Scholar, A. Darst S. et bacterial sigma factor a with DNA Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the of RNAP a DNA K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex.Science. 2002; 296: 1285-1290Crossref PubMed Scopus (543) Google Scholar, J. J. A. et of bacterial RNA polymerase and the RNA open 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In σA, the region is that deletion of between 132-179 σA of residues in regions the we that the a role to phosphorylation regulates many in including of is the σ factors and its factor are of and the phosphorylation of with interactions of the response by an essential protein in Mycobacterium S. A. 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