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.


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  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, 210218 [View Article][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, 397404 [View Article][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, 180200 [View Article][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, 566582 [View Article][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, 205212 [View Article][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, 12371249 [View Article][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, 270273 [View Article][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, 1119 [View Article][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, 462468 [View Article][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, 5766 [View Article][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, 6770.[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, 167179 [View Article][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, 214228 [View Article][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, 13071322 [View Article][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, 67736782[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, 643657 [View Article][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, 56285633.[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, 621632 [View Article][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, 503522 [View Article][PubMed] [Google Scholar]
  20. Hett E. C., Rubin E. J. (2008). Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72, 126156 [View Article][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, 658668 [View Article][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 [View Article][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 [View Article][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, 140147 [View Article][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, 3950 [View Article][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, 672684 [View Article][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, 699707.[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, 420438 [View Article][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, 348364 [View Article][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, 2388723899 [View Article][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, 41204129 [View Article][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, 1581615827 [View Article][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, 12741286.[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, 42694281 [View Article][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, 605626 [View Article][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, 20742086 [View Article][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, 29852995 [View Article][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), S128S135 [View Article][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, 4752.[CrossRef] [Google Scholar]
  41. van Kessel J. C., Hatfull G. F. (2007). Recombineering in Mycobacterium tuberculosis . Nat Methods 4, 147152 [View Article][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, 15911594 [View Article][PubMed] [Google Scholar]
  43. Zahrt T. C., Deretic V. (2000). An essential two-component signal transduction system in Mycobacterium tuberculosis . J Bacteriol 182, 38323838 [View Article][PubMed] [Google Scholar]

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