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Volume 167, Issue 10

DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the operon of Free

Abstract

MSMEG_2295 is a TetR family protein encoded by the first gene of a (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the operon and several other genes. Using a knockout of , we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in although in the complemented strain, it was high. We also show that knocking out of causes resistance to HO, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.

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  • Accepted:
  • Published Online:
Keyword(s): dinB2 , MSMEG_0089 , MSMEG_2295 , pyruvate metabolism and repressor
Funding
This study was supported by the:
  • bose institute
    • Principle Award Recipient: PoulamiGhosh
  • council of scientific and industrial research, india (Award 09/015(0491)2016-EMR-I)
    • Principle Award Recipient: MadhuManti Patra
  • dst wosa
    • Principle Award Recipient: ShreyaSengupta
© 2021 The Authors
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2021-10-19
2024-04-25
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References

  1. Tiberi S, Migliori GB, Muhwa Chakaya J, Kaesava T, Al Abri SS et al. Commemorating World TB Day 2020: “IT’S TIME” - It’s time to End the Global TB Epidemic. Int J Infect Dis 2020; 92S:S4S1
     [Google Scholar]
  2. Nasiri MJ, Haeili M, Ghazi M, Goudarzi H, Pormohammad A et al. New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria. Front Microbiol 2017; 8:681 [View Article] [PubMed]
     [Google Scholar]
  3. Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol 2007; 5:48–56 [View Article] [PubMed]
     [Google Scholar]
  4. Perron GG, Hall AR, Buckling A. Hypermutability and compensatory adaptation in antibiotic-resistant bacteria. Am Nat 2010; 176:303–311 [View Article] [PubMed]
     [Google Scholar]
  5. Varhimo E, Savijoki K, Jefremoff H, Jalava J, Sukura A et al. Ciprofloxacin induces mutagenesis to antibiotic resistance independent of UmuC in Streptococcus uberis. Environ Microbiol 2008; 10:2179–2183 [View Article] [PubMed]
     [Google Scholar]
  6. Jarosz DF, Beuning PJ, Cohen SE, Walker GC. Y-family DNA polymerases in Escherichia coli. Trends Microbiol 2007; 15:70–77 [View Article] [PubMed]
     [Google Scholar]
  7. Singh A. Guardians of the mycobacterial genome: A review on DNA repair systems in Mycobacterium tuberculosis. Microbiology (Reading) 2017; 163:1740–1758 [View Article] [PubMed]
     [Google Scholar]
  8. Sale JE, Lehmann AR, Woodgate R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nat Rev Mol Cell Biol 2012; 13:141–152 [View Article] [PubMed]
     [Google Scholar]
  9. Witkin EM. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 1976; 40:869–907 [View Article] [PubMed]
     [Google Scholar]
  10. Miller C, Thomsen LE, Gaggero C, Mosseri R, Ingmer H et al. SOS response induction by beta-lactams and bacterial defense against antibiotic lethality. Science 2004; 305:1629–1631 [View Article] [PubMed]
     [Google Scholar]
  11. Ordonez H, Uson ML, Shuman S. Characterization of three mycobacterial DinB (DNA polymerase IV) paralogs highlights DinB2 as naturally adept at ribonucleotide incorporation. Nucleic Acids Res 2014; 42:11056–11070 [View Article] [PubMed]
     [Google Scholar]
  12. Boshoff HI, Reed MB, Barry CE 3rd, Mizrahi V. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 2003; 113:183–193 [View Article] [PubMed]
     [Google Scholar]
  13. Kana BD, Abrahams GL, Sung N, Warner DF, Gordhan BG et al. Role of the DinB homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis. J Bacteriol 2010; 192:2220–2227 [View Article] [PubMed]
     [Google Scholar]
  14. Ghosh S, Samaddar S, Kirtania P, Das Gupta SK. A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene. J Bacteriol 2016; 198:352–362 [View Article] [PubMed]
     [Google Scholar]
  15. Balhana RJ, Singla A, Sikder MH, Withers M, Kendall SL. Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions. BMC genomics 2015; 16:479 [View Article] [PubMed]
     [Google Scholar]
  16. Rosenberg AH, Lade BN, Chui DS, Lin SW, Dunn JJ et al. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1987; 56:125–135 [View Article] [PubMed]
     [Google Scholar]
  17. Warner DF, Etienne G, Wang XM, Matsoso LG, Dawes SS et al. A derivative of Mycobacterium smegmatis mc(2)155 that lacks the duplicated chromosomal region. Tuberculosis (Edinb) 2006; 86:438–444 [View Article] [PubMed]
     [Google Scholar]
  18. van Kessel JC, Hatfull GF. Recombineering in Mycobacterium tuberculosis. Nat Methods 2007; 4:147–152 [View Article] [PubMed]
     [Google Scholar]
  19. Parish T, Stoker NG. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 2000; 146:1969–1975 [View Article] [PubMed]
     [Google Scholar]
  20. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 2016; 33:1870–1874 [View Article] [PubMed]
     [Google Scholar]
  21. Grant CE, Johnson J, Bailey TL, Noble WS. MCAST: scanning for cis-regulatory motif clusters. Bioinformatics 2016; 32:1217–1219 [View Article] [PubMed]
     [Google Scholar]
  22. Sarkar A, Ghosh S, Shaw R, Patra MM, Calcuttawala F et al. Mycobacterium tuberculosis thymidylate synthase (ThyX) is a target for plumbagin, a natural product with antimycobacterial activity. PloS one 2020; 15:e0228657 [View Article] [PubMed]
     [Google Scholar]
  23. Basu A, Chawla-Sarkar M, Chakrabarti S, Das Gupta SK. Origin binding activity of the Mycobacterial plasmid pAL5000 replication protein RepB is stimulated through interactions with host factors and coupled expression of repA. J Bacteriol 2002; 184:2204–2214 [View Article] [PubMed]
     [Google Scholar]
  24. Chatterjee S, Basu A, Basu A, Das Gupta SK. DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex. J Bacteriol 2007; 189:8584–8592 [View Article] [PubMed]
     [Google Scholar]
  25. Bhawsinghka N, Dutta A, Mukhopadhyay J, Das Gupta SK. A transcriptomic analysis of the mycobacteriophage D29 genome reveals the presence of novel stoperator-associated promoters in its right arm. Microbiology 2018; 164:1168–1179 [View Article] [PubMed]
     [Google Scholar]
  26. Soo PC, Horng YT, Lai MJ, Wei JR, Hsieh SC et al. Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity. J Bacteriol 2007; 189:109–118 [View Article] [PubMed]
     [Google Scholar]
  27. Martini MC, Zhou Y, Sun H, Shell SS. Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia. Front Microbiol 2019; 10:591 [View Article] [PubMed]
     [Google Scholar]
  28. Tala A, Damiano F, Gallo G, Pinatel E, Calcagnile M et al. Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces. Metab Eng 2018; 48:254–268 [View Article] [PubMed]
     [Google Scholar]
  29. de Kok A, Hengeveld AF, Martin A, Westphal AH. The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1998; 1385:353–366 [View Article]
     [Google Scholar]
  30. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797–810 [View Article] [PubMed]
     [Google Scholar]
  31. Alguel Y, Meng C, Teran W, Krell T, Ramos JL et al. Crystal structures of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials. J Mol Biol 2007; 369:829–840 [View Article] [PubMed]
     [Google Scholar]
  32. Daniels C, Daddaoua A, Lu D, Zhang X, Ramos JL. Domain cross-talk during effector binding to the multidrug binding TTGR regulator. J Biol Chem 2010; 285:21372–21381 [View Article] [PubMed]
     [Google Scholar]
  33. Boshoff HI, Myers TG, Copp BR, McNeil MR, Wilson MA et al. 3rd. The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 279:40174–40184 [View Article] [PubMed]
     [Google Scholar]
  34. Dwyer DJ, Kohanski MA, Collins JJ. Role of reactive oxygen species in antibiotic action and resistance. Curr Opin Microbiol 2009; 12:482–489 [View Article] [PubMed]
     [Google Scholar]
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DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the dinB2 operon of Mycobacterium smegmatis
Microbiology 167, 001097 (2021); https://doi.org/10.1099/mic.0.001097
/content/journal/micro/10.1099/mic.0.001097
/content/journal/micro/10.1099/mic.0.001097
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