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.
Anirudh K Singh, Debashis Dutta, Vandana Singh, Vishal Srivastava, Rajesh K Biswas, Bhupendra N Singh
Author Information
Anirudh K Singh: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
Debashis Dutta: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
Vandana Singh: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
Vishal Srivastava: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
Rajesh K Biswas: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
Bhupendra N Singh: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, 226031, India.
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 mc(2) 155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoidpigmentation which rendered increased susceptibility to H2 O2 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 mc(2) 155 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.
