MSMEG_5850, a stress-induced TetR protein, involved in global transcription regulation in Mycobacterium smegmatis
Abstract
Aim: To decipher the role of MSMEG_5850 in the physiology of mycobacteria. Methods:MSMEG_5850 was knocked out and RNA sequencing was performed. MSMEG_5850 protein was purified from the Escherichia coli pET28a system. Electrophoretic mobility shift assay and size exclusion chromatography were used to determine the binding of MSMEG_5850 to its motif and binding stoichiometry. The effect of nutritional stress was monitored. Results: Transcriptome analysis revealed the differential expression of 148 genes in an MSMEG_5850 knockout strain. MSMEG_5850 had control over 50 genes because those genes had a binding motif upstream of their sequence. The electrophoretic mobility shift assay showed MSMEG_5850 bound to its motif as a monomer. MSMEG_5850 was upregulated under nutritional stress and promoted the survival of mycobacteria. Conclusion: The study confirms the role of MSMEG_5850 in global transcriptional regulation.
Graphical abstract
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References
- 1. . Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions. BMC Genomics 16(1), 479 (2015).
- 2. . Structural basis of gene regulation by the tetracycline inducible Tet repressor–operator system. Nat. Struct. Biol. 7(3), 215–219 (2000).
- 3. . VceR negatively regulates the vceCAB MDR efflux operon and positively regulates its own synthesis in Vibrio cholerae 569B. Can. J. Microbiol. 53(7), 888–900 (2007).
- 4. . Conditionally positive effect of the TetR-family transcriptional regulator AtrA on streptomycin production by Streptomyces griseus. Microbiology 154(3), 905–914 (2008).
- 5. Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon. Mol. Microbiol. 58(2), 580–595 (2005).
- 6. . A TetR family transcriptional factor directly regulates the expression of a 3-methyladenine DNA glycosylase and physically interacts with the enzyme to stimulate its base excision activity in Mycobacterium bovis BCG. J. Biol. Chem. 289(13), 9065–9075 (2014).
- 7. . The TetR family of regulators. Microbiol. Mol. Biol. Rev. 77(3), 440–475 (2013).
- 8. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 69(2), 326–356 (2005).
- 9. A survey of best practices for RNA-seq data analysis. Genome Biol. 17(1), 13(2016). • Describes different methods of RNA seq data analysis.
- 10. Transcriptional profile of Mycobacterium tuberculosis in an in vitro model of intraocular tuberculosis. Front. Cell. Infect. Microbiol. 8, 330 (2018).
- 11. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 50(W1), W276–W279 (2022). •• Multiple sequence alignment.
- 12. . In the box. Ragtrader (Mar 2020), 12–13 (2020). •• Motif analysis by MEME tool.
- 13. . Mycobacteria Protocols. Springer, Humana New York, NY, USA (1998). • Describes the protocol for preparation of electrocompetent cells of mycobacteria.
- 14. . Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4), 402–408 (2001). • Relative quantification of genes.
- 15. Metabolomics studies to decipher stress responses in Mycobacterium smegmatis point to a putative pathway of methylated amine biosynthesis. J. Bacteriol. 201(15), e00707–00718 (2019).
- 16. . TetR-family transcription factors in Gram-negative bacteria: conservation, variation and implications for efflux-mediated antimicrobial resistance. BMC Genomics 20(1), 731 (2019).
- 17. Structural and functional analysis of the transcriptional regulator Rv3066 of Mycobacterium tuberculosis. Nucleic Acids Res. 40(18), 9340–9355 (2012).
- 18. bkaR is a TetR-type repressor that controls an operon associated with branched-chain keto-acid metabolism in Mycobacteria. FEMS Microbiol. Lett. 345(2), 132–140 (2013).
- 19. Tuning a nitrate reductase for function: the first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties. J. Biol. Chem. 279(31), 32212–32218 (2004).
- 20. . A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase. Plant Mol. Biol. 30(4), 845–850 (1996).
- 21. . Deciphering the metabolic response of Mycobacterium tuberculosis to nitrogen stress. Mol. Microbiol. 97(6), 1142–1157 (2015).
- 22. . Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra. Mol. Biol. Rep. 49(6), 4583–4593 (2022).
- 23. Developmental transcriptome of resting cell formation in Mycobacterium smegmatis. BMC Genomics 17(1), 837 (2016).
- 24. . Genome context as a predictive tool for identifying regulatory targets of the TetR family transcriptional regulators. PLOS ONE 7(11), e50562 (2012). •• Defines the different types of TetR family transcriptional regulators.
- 25. . Global analysis of gene transcription regulation in prokaryotes. Cell. Mol. Life Sci. C 63(19), 2260–2290 (2006).
- 26. . The σ factors of Mycobacterium tuberculosis. FEMS Microbiol. Rev. 30(6), 926–941 (2006). • Induction of different σ factors under different stresses.
- 27. . The sigma factors of Mycobacterium tuberculosis: regulation of the regulators. FEBS J. 277(3), 605–626 (2010). • Induction of different σ factors under different stresses.