Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

Abstract

In Mycobacterium smegmatis, sigF is widely expressed during different growth stages and plays role in adaptation to stationary phase and oxidative stress. Using a sigF deletion mutant of M. smegmatis mc2155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoid pigmentation which rendered increased susceptibility to H2O2 induced oxidative stress in M. smegmatis. SigF modulates the cell surface architecture and lipid biosynthesis extending the repertoire of SigF function in this species. M. smegmatis SigF regulon included variety of genes expressed during exponential and stationary phases of growth and those responsible for oxidative stress, lipid biosynthesis, energy, and central intermediary metabolism. Furthermore, we report the identification of a SigF antagonist, an anti‐sigma factor (RsbW), which upon overexpression in M. smegmatis wild type strain produced a phenotype similar to M. smegmatis mc2155 ΔsigF strain. The SigF‐anti‐SigF interaction is duly validated using bacterial two‐hybrid and pull down assays. In addition, anti‐sigma factor antagonists, RsfA and RsfB were identified and their interactions with anti‐sigma factor were experimentally validated. Identification of these proteins will help decode regulatory circuit of this alternate sigma factor.

Roles of three FurA paralogs in the regulation of genes pertaining to peroxide defense in Mycobacterium smegmatis mc2155

“…Interestingly, a large portion (29 genes, 78.4%) of 37 repressed genes overlaps the known SigF regulon of M. smegmatis (Fig. and Supporting Information Table S2) (Singh et al ., ). The significantly repressed genes other than the SigF regulon and the genes of unknown function include aceA ( MSMEG_0911 ) encoding isocitrate lyase (McKinney et al ., ; Munoz‐Elias and McKinney, ), as well as glpK ( MSMEG_6759 ) and glpD ( MSMEG_6761 ) encoding glycerol kinase and glycerol‐3‐phosphate dehydrogenase, respectively (Seno and Chater, ; Titgemeyer et al ., ), implying downregulation of the glyoxylate shunt and glycerol metabolism in the Δ f1f2f3 mutant.…”

“…SigF is known to be involved in cell surface stress (heat, SDS, antibiotics, etc.) and oxidative stress responses, as well as the adaptation to stationary growth phase (Provvedi et al ., ; Humpel et al ., ; Singh et al ., ). This finding implies that the genes of the SigF regulon are positively regulated by FurA in an indirect way.…”

Structural insights ofMycobacteriumGTPase-Obg and anti-sigma-F factor Usfx interaction

“…14 β-sheets (numbered [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Similarly, Usfx secondary structure is characterized by 3 α-helices (numbered i-iii) and 5 β-sheets (numbered i-v) (Table S2).…”

“…SigF is another sigma factor, which is considered a stress response factor as its expression increases in MTB under stress conditions, such as nutrient starvation, cold shock, and antibiotics . In another study, cell wall–associated proteins were found differentially regulated after over expression of SigF in MTB . The activity of sigma factors is controlled by sigma factor antagonists called anti‐sigma factors, which bind with sigma factors and prevent their interaction with RNAP and thus, preventing expression of genes controlled by that specific sigma factor.…”

“…7 In another study, cell wall-associated proteins were found differentially regulated after over expression of SigF in MTB. 8 The activity of sigma factors is controlled by sigma factor antagonists called anti-sigma factors, which bind with sigma factors and prevent their interaction with RNAP and thus, preventing expression of genes controlled by that specific sigma factor. After receiving signals from external stimuli, anti-sigma factor releases sigma factor, resulting in facilitation of gene transcription.…”