1887

Abstract

The resuscitation-promoting factors of are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of , is required for reactivation of from chronic infection , underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of , and suggested from promoter-driven GFP expression and transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that transcription is regulated by MtrA – the response regulator of the essential two-component system MtrAB. Association of MtrA with the promoter region was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of using a biotinylated DNA fragment encompassing the MtrA-binding site on the promoter, confirming that MtrA binds to the promoter. Enhanced GFP fluorescence driven by the promoter, upon deletion of the MtrA-binding site, and repression of expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the promoter in the presence of MtrA. transcription assays confirmed that MtrA inhibits RNAP-driven transcription.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000087
2015-06-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/161/6/1271.html?itemId=/content/journal/micro/10.1099/mic.0.000087&mimeType=html&fmt=ahah

References

  1. Al Zayer M. , Stankowska D. , Dziedzic R. , Sarva K. , Madiraju M. V. , Rajagopalan M. . ( 2011;). Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth. . Plasmid 65:, 210–218 [CrossRef] [PubMed]
    [Google Scholar]
  2. Banerjee R. , Rudra P. , Prajapati R. K. , Sengupta S. , Mukhopadhyay J. . ( 2014;). Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification. . Tuberculosis (Edinb) 94:, 397–404 [CrossRef] [PubMed]
    [Google Scholar]
  3. Bisicchia P. , Noone D. , Lioliou E. , Howell A. , Quigley S. , Jensen T. , Jarmer H. , Devine K. M. . ( 2007;). The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis . . Mol Microbiol 65:, 180–200 [CrossRef] [PubMed]
    [Google Scholar]
  4. Bretl D. J. , Demetriadou C. , Zahrt T. C. . ( 2011;). Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis . . Microbiol Mol Biol Rev 75:, 566–582 [CrossRef] [PubMed]
    [Google Scholar]
  5. Brocker M. , Bott M. . ( 2006;). Evidence for activator and repressor functions of the response regulator MtrA from Corynebacterium glutamicum . . FEMS Microbiol Lett 264:, 205–212 [CrossRef] [PubMed]
    [Google Scholar]
  6. Brocker M. , Mack C. , Bott M. . ( 2011;). Target genes, consensus binding site, and role of phosphorylation for the response regulator MtrA of Corynebacterium glutamicum . . J Bacteriol 193:, 1237–1249 [CrossRef] [PubMed]
    [Google Scholar]
  7. Cohen-Gonsaud M. , Barthe P. , Bagnéris C. , Henderson B. , Ward J. , Roumestand C. , Keep N. H. . ( 2005;). The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes. . Nat Struct Mol Biol 12:, 270–273 [CrossRef] [PubMed]
    [Google Scholar]
  8. Cousin C. , Derouiche A. , Shi L. , Pagot Y. , Poncet S. , Mijakovic I. . ( 2013;). Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation. . FEMS Microbiol Lett 346:, 11–19 [CrossRef] [PubMed]
    [Google Scholar]
  9. Davies A. P. , Dhillon A. P. , Young M. , Henderson B. , McHugh T. D. , Gillespie S. H. . ( 2008;). Resuscitation-promoting factors are expressed in Mycobacterium tuberculosis-infected human tissue. . Tuberculosis (Edinb) 88:, 462–468 [CrossRef] [PubMed]
    [Google Scholar]
  10. Deng L. L. , Humphries D. E. , Arbeit R. D. , Carlton L. E. , Smole S. C. , Carroll J. D. . ( 2005;). Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis . . Biochim Biophys Acta 1747:, 57–66 [CrossRef] [PubMed]
    [Google Scholar]
  11. Doukhan L. , Predich M. , Nair G. , Dussurget O. , Mandic-Mulec I. , Cole S. T. , Smith D. R. , Smith I. . ( 1995;) Genomic organization of the mycobacterial sigma gene cluster. . Gene 165:, 67–70.[CrossRef]
    [Google Scholar]
  12. Downing K. J. , Betts J. C. , Young D. I. , McAdam R. A. , Kelly F. , Young M. , Mizrahi V. . ( 2004;). Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy. . Tuberculosis (Edinb) 84:, 167–179 [CrossRef] [PubMed]
    [Google Scholar]
  13. Dubrac S. , Msadek T. . ( 2008;). Tearing down the wall: peptidoglycan metabolism and the WalK/WalR (YycG/YycF) essential two-component system. . Adv Exp Med Biol 631:, 214–228 [CrossRef] [PubMed]
    [Google Scholar]
  14. Dubrac S. , Bisicchia P. , Devine K. M. , Msadek T. . ( 2008;). A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway. . Mol Microbiol 70:, 1307–1322 [CrossRef] [PubMed]
    [Google Scholar]
  15. Engel H. , Kazemier B. , Keck W. . ( 1991;). Murein-metabolizing enzymes from Escherichia coli: sequence analysis and controlled overexpression of the slt gene, which encodes the soluble lytic transglycosylase. . J Bacteriol 173:, 6773–6782 [PubMed]
    [Google Scholar]
  16. Fol M. , Chauhan A. , Nair N. K. , Maloney E. , Moomey M. , Jagannath C. , Madiraju M. V. , Rajagopalan M. . ( 2006;). Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator. . Mol Microbiol 60:, 643–657 [CrossRef] [PubMed]
    [Google Scholar]
  17. Fontán P. A. , Voskuil M. I. , Gomez M. , Tan D. , Pardini M. , Manganelli R. , Fattorini L. , Schoolnik G. K. , Smith I. . ( 2009;) The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth. . J Bacteriol 191:, 5628–5633.[CrossRef]
    [Google Scholar]
  18. Fukushima T. , Szurmant H. , Kim E. J. , Perego M. , Hoch J. A. . ( 2008;). A sensor histidine kinase co-ordinates cell wall architecture with cell division in Bacillus subtilis . . Mol Microbiol 69:, 621–632 [CrossRef] [PubMed]
    [Google Scholar]
  19. Fukushima T. , Furihata I. , Emmins R. , Daniel R. A. , Hoch J. A. , Szurmant H. . ( 2011;). A role for the essential YycG sensor histidine kinase in sensing cell division. . Mol Microbiol 79:, 503–522 [CrossRef] [PubMed]
    [Google Scholar]
  20. Hett E. C. , Rubin E. J. . ( 2008;). Bacterial growth and cell division: a mycobacterial perspective. . Microbiol Mol Biol Rev 72:, 126–156 [CrossRef] [PubMed]
    [Google Scholar]
  21. Hett E. C. , Chao M. C. , Steyn A. J. , Fortune S. M. , Deng L. L. , Rubin E. J. . ( 2007;). A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis . . Mol Microbiol 66:, 658–668 [CrossRef] [PubMed]
    [Google Scholar]
  22. Hett E. C. , Chao M. C. , Deng L. L. , Rubin E. J. . ( 2008;). A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor. . PLoS Pathog 4:, e1000001 [CrossRef] [PubMed]
    [Google Scholar]
  23. Hett E. C. , Chao M. C. , Rubin E. J. . ( 2010;). Interaction and modulation of two antagonistic cell wall enzymes of mycobacteria. . PLoS Pathog 6:, e1001020 [CrossRef] [PubMed]
    [Google Scholar]
  24. Jacques J. -F. , Rodrigue S. , Brzezinski R. , Gaudreau L. . ( 2006;). A recombinant Mycobacterium tuberculosis in vitro transcription system. . FEMS Microbiol Lett 255:, 140–147 [CrossRef] [PubMed]
    [Google Scholar]
  25. Kana B. D. , Mizrahi V. . ( 2010;). Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. . FEMS Immunol Med Microbiol 58:, 39–50 [CrossRef] [PubMed]
    [Google Scholar]
  26. Kana B. D. , Gordhan B. G. , Downing K. J. , Sung N. , Vostroktunova G. , Machowski E. E. , Tsenova L. , Young M. , Kaprelyants A. , other authors . ( 2008;). The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro . . Mol Microbiol 67:, 672–684 [CrossRef] [PubMed]
    [Google Scholar]
  27. Lee J. H. , Karakousis P. C. , Bishai W. R. . ( 2008;) Roles of SigB and SigF in the Mycobacterium tuberculosis sigma factor network. . J Bacteriol 190:, 699–707.[CrossRef]
    [Google Scholar]
  28. Miller J. H. . ( 1992;). A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria . Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  29. Möker N. , Brocker M. , Schaffer S. , Krämer R. , Morbach S. , Bott M. . ( 2004;). Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. . Mol Microbiol 54:, 420–438 [CrossRef] [PubMed]
    [Google Scholar]
  30. Nguyen H. T. , Wolff K. A. , Cartabuke R. H. , Ogwang S. , Nguyen L. . ( 2010;). A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium . Mol Microbiol 76:, 348–364 [CrossRef] [PubMed]
    [Google Scholar]
  31. Plocinska R. , Purushotham G. , Sarva K. , Vadrevu I. S. , Pandeeti E. V. , Arora N. , Plocinski P. , Madiraju M. V. , Rajagopalan M. . ( 2012;). Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression. . J Biol Chem 287:, 23887–23899 [CrossRef] [PubMed]
    [Google Scholar]
  32. Plocinska R. , Martinez L. , Gorla P. , Pandeeti E. , Sarva K. , Blaszczyk E. , Dziadek J. , Madiraju M. V. , Rajagopalan M. . ( 2014;). Mycobacterium tuberculosis MtrB sensor kinase interactions with FtsI and Wag31 proteins reveal a role for MtrB distinct from that regulating MtrA activities. . J Bacteriol 196:, 4120–4129 [CrossRef] [PubMed]
    [Google Scholar]
  33. Rajagopalan M. , Dziedzic R. , Al Zayer M. , Stankowska D. , Ouimet M. C. , Bastedo D. P. , Marczynski G. T. , Madiraju M. V. . ( 2010;). Mycobacterium tuberculosis origin of replication and the promoter for immunodominant secreted antigen 85B are the targets of MtrA, the essential response regulator. . J Biol Chem 285:, 15816–15827 [CrossRef] [PubMed]
    [Google Scholar]
  34. Rickman L. , Scott C. , Hunt D. M. , Hutchinson T. , Menéndez M. C. , Whalan R. , Hinds J. , Colston M. J. , Green J. . & other authors. ( 2005;). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. . Mol Microbiol. 56:, 1274–1286.[CrossRef]
    [Google Scholar]
  35. Russell-Goldman E. , Xu J. , Wang X. , Chan J. , Tufariello J. M. . ( 2008;). A Mycobacterium tuberculosis Rpf double-knockout strain exhibits profound defects in reactivation from chronic tuberculosis and innate immunity phenotypes. . Infect Immun 76:, 4269–4281 [CrossRef] [PubMed]
    [Google Scholar]
  36. Sachdeva P. , Misra R. , Tyagi A. K. , Singh Y. . ( 2010;). The sigma factors of Mycobacterium tuberculosis: regulation of the regulators. . FEBS J 277:, 605–626 [CrossRef] [PubMed]
    [Google Scholar]
  37. Sanyal S. , Banerjee S. K. , Banerjee R. , Mukhopadhyay J. , Kundu M. . ( 2013;). Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3-RegX3 and the extracytoplasmic function sigma factor, sigma E. . Microbiology 159:, 2074–2086 [CrossRef] [PubMed]
    [Google Scholar]
  38. Tufariello J. M. , Mi K. , Xu J. , Manabe Y. C. , Kesavan A. K. , Drumm J. , Tanaka K. , Jacobs W. R. Jr , Chan J. . ( 2006;). Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis. . Infect Immun 74:, 2985–2995 [CrossRef] [PubMed]
    [Google Scholar]
  39. Vadrevu I. S. , Lofton H. , Sarva K. , Blasczyk E. , Plocinska R. , Chinnaswamy J. , Madiraju M. , Rajagopalan M. . ( 2011;). ChiZ levels modulate cell division process in mycobacteria. . Tuberculosis (Edinb) 91: (Suppl 1), S128–S135 [CrossRef] [PubMed]
    [Google Scholar]
  40. Valdivia R. H. , Hromockyj A. E. , Monack D. , Ramakrishnan L. , Falkow S. . ( 1996;). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. . Gene 173:, 47–52.[CrossRef]
    [Google Scholar]
  41. van Kessel J. C. , Hatfull G. F. . ( 2007;). Recombineering in Mycobacterium tuberculosis . . Nat Methods 4:, 147–152 [CrossRef] [PubMed]
    [Google Scholar]
  42. Wivagg C. N. , Hung D. T. . ( 2012;). Resuscitation-promoting factors are required for β-lactam tolerance and the permeability barrier in Mycobacterium tuberculosis . . Antimicrob Agents Chemother 56:, 1591–1594 [CrossRef] [PubMed]
    [Google Scholar]
  43. Zahrt T. C. , Deretic V. . ( 2000;). An essential two-component signal transduction system in Mycobacterium tuberculosis . . J Bacteriol 182:, 3832–3838 [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000087
Loading
/content/journal/micro/10.1099/mic.0.000087
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error