1887

Abstract

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of , were cloned and the protein products were overexpressed, purified and refolded as N-terminally His-tagged proteins from . DevS underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His and Asp as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp and Asp residues, postulated to form the acidic Mg-binding pocket, and the invariant Lys of DevR, abrogated phosphoryl transfer from DevS to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the operon was induced at the RNA level in hypoxic cultures of H37Rv and was associated with an increase in the level of DevR protein. However, in a mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

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2004-04-01
2024-03-29
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References

  1. Bagchi G., Mayuri Tyagi J. S. 2003; Hypoxia-responsive expression of Mycobacterium tuberculosis Rv3134c and devR promoters in Mycobacterium smegmatis. Microbiology 149:2303–2305 [CrossRef]
    [Google Scholar]
  2. Boon C., Dick T. 2002; Mycobacterium bovis BCG response regulator essential for hypoxic dormancy. J Bacteriol 184:6760–6767 [CrossRef]
    [Google Scholar]
  3. Boon C., Li R., Qi R., Dick T. 2001; Proteins of Mycobacterium bovis BCG induced in Wayne dormancy model. J Bacteriol 182:2672–2676
    [Google Scholar]
  4. Cole S. T., Brosch R., Parkhill J.39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
    [Google Scholar]
  5. Dale J. W., Patki A. 1990; Mycobacterial gene expression and regulation. In Molecular Biology of Mycobacteria pp. 173–198 Edited by McFadden J. Guildford, UK: Surrey University Press;
    [Google Scholar]
  6. Dasgupta N., Kapur V., Singh K. K., Das T. K., Sachdeva S., Jyothisri K., Tyagi J. S. 2000; Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. Tuber Lung Dis 80:141–159 [CrossRef]
    [Google Scholar]
  7. Delgado-Nixon V. M., Gonzalez G., Gilles-Gonzalez M. A. 2000; Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor. Biochemistry 39:2685–2691 [CrossRef]
    [Google Scholar]
  8. Emmerich R., Panglungtshang K., Strehler P., Hennecke H., Fischer H. M. 1999; Phosphorylation, dephosphorylation and DNA-binding of Bradyrhizobium japonicum RegSR two-component regulatory proteins. Eur J Biochem 263:455–463 [CrossRef]
    [Google Scholar]
  9. Florczyk M. A., McCue L. A., Purkayastha A., Currenti E., Wolin M. J., McDonough K. A. 2003; A family of acr-coregulated Mycobacterium tuberculosis genes shares a common DNA motif and requiresRv3133c (dosR or devR) for expression. Infect Immun 71:5332–5343 [CrossRef]
    [Google Scholar]
  10. Fujita M., Losick R. 2003; The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division. Genes Dev 17:1166–1174 [CrossRef]
    [Google Scholar]
  11. Georgellis D., Kwon O., Lin E. C. 1999; Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites. An in vitro study with different protein modules. J Biol Chem 274:35950–35954 [CrossRef]
    [Google Scholar]
  12. Haydel S. E., Dunlap N. E., Benjamin W. H., Jr. 1999; In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TrcR/TrcS proteins. Microb Pathog 26:195–206 [CrossRef]
    [Google Scholar]
  13. Himpens S., Locht C., Supply P. 2000; Molecular characterization of the mycobacterial SenX3-RegX3 two-component system: evidence for autoregulation. Microbiology 146:3091–3098
    [Google Scholar]
  14. Ho Y. S., Burden L. M., Hurley J. H. 2000; Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor. EMBO J 19:5288–5299 [CrossRef]
    [Google Scholar]
  15. Hou S., Freitas T., Larsen R. W.7 other authors 2001; Globin-coupled sensors: a class of heme-containing sensors in Archaea and Bacteria. Proc Natl Acad Sci U S A 98:9353–9398 [CrossRef]
    [Google Scholar]
  16. Kiley P. J., Beinert H. 1998; Oxygen sensing by global regulator, FNR – the role of the iron–sulfur cluster. FEMS Microbiol Rev 22:341–352 [CrossRef]
    [Google Scholar]
  17. Kinger A. K., Tyagi J. S. 1993; Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis. Gene 131:113–117 [CrossRef]
    [Google Scholar]
  18. Malhotra V., Sharma D., Ramanathan V. D.8 other authors 2004; Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 231:237–245 [CrossRef]
    [Google Scholar]
  19. Mayuri, Bagchi G., Das T. K., Tyagi J. S. 2002; Molecular analysis of the dormancy response in Mycobacterium smegmatis: expression analysis of genes encoding the DevR-DevS two-component system, Rv3134c and chaperone alpha-crystallin homologues. FEMS Microbiol Lett 211:231–237
    [Google Scholar]
  20. Mukai K., Kawata M., Tanaka T. 1990; Isolation and phosphorylation of the Bacillus subtilis degS and degU gene products. J Biol Chem 265:20000–20006
    [Google Scholar]
  21. Park H. D., Guinn K. M., Harrell M. I., Liao R., Voskuil M. I., Tompa M., Schoolnik G. K., Sherman D. R. 2003; Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48:833–843 [CrossRef]
    [Google Scholar]
  22. Parkinson J. S., Kofoid E. C. 1992; Communication modules in bacterial signalling proteins. Annu Rev Genet 26:71–112 [CrossRef]
    [Google Scholar]
  23. Patschkowski T., Schluter A., Priefer U. B. 1996; Rhizobium legumisosarum bv.viciae contains a second fnr/fixK-like gene and an unusual fixL homologue. Mol Microbiol 21:267–80 [CrossRef]
    [Google Scholar]
  24. Saini D. K., Pant N., Das T. K., Tyagi J. S. 2002; Cloning, overexpression, purification and matrix-assisted refolding of DevS (Rv3132c) histidine protein kinase of Mycobacterium tuberculosis. Protein Expr Purif 25:203–208 [CrossRef]
    [Google Scholar]
  25. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  26. Sherman D. R., Voskuil M., Schnappinger D., Liao R., Harrell M. I., Schoolnik G. K. 2001; Regulation of the Mycobacterium tuberculosis hypoxia response gene encoding α-crystallin. Proc Natl Acad Sci U S A 98:7534–7539 [CrossRef]
    [Google Scholar]
  27. Stock J. B., Surette M. G., Levit M., Park P. 1995; Two-component signal transduction systems: structure-function relationships and mechanisms of catalysis. In Two-Component Signal Transduction pp. 25–51 Edited by Hoch J. A., Silhavy T. J. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  28. Stock A. M., Robinson V. L., Goudreau P. N. 2000; Two-component signal transduction. Annu Rev Biochem 69:183–215 [CrossRef]
    [Google Scholar]
  29. Voskuil M. I., Schnappinger D., Visconti K. C., Harrell M. I., Dolganov G. M., Sherman D. R., Schoolnik G. K. 2003; Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198:705–713 [CrossRef]
    [Google Scholar]
  30. Walker M. S., DeMoss J. A. 1993; Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system. NarX and NarL. J Biol Chem 268:8391–8393
    [Google Scholar]
  31. Wayne L. G., Hayes L. G. 1996; An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64:2062–2069
    [Google Scholar]
  32. Zahrt T. C., Wozniak C., Jones D., Trevett A. 2003; Functional analysis of the Mycobacterium tuberculosis MprAB two-component signal transduction system. Infect Immun 71:6962–6970 [CrossRef]
    [Google Scholar]
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