생명과학회지 (Journal of Life Science)

  • 제20권6호
  • /
  • Pages.853-860
  • /
  • 2010
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Subunit 간의 disulfide 결합 형성에 의한 Mycobacterium smegmatis DevS histidine kinase의 불활성화

Inactivation of the DevS Histidine Kinase of Mycobacterium smegmatis by the Formation of the Intersubunit Disulfide Bond

  • 이진목 (부산대학교 미생물학과) ;
  • 박광진 (부산대학교 미생물학과) ;
  • 김민주 (부산대학교 미생물학과) ;
  • 고인정 (KAIST 부설 한국영재학교) ;
  • 오정일 (부산대학교 미생물학과)
  • Lee, Jin-Mok (Department of Microbiology, Pusan National University) ;
  • Park, Kwang-Jin (Department of Microbiology, Pusan National University) ;
  • Kim, Min-Ju (Department of Microbiology, Pusan National University) ;
  • Ko, In-Jeong (Korea Science Academy of KAIST) ;
  • Oh, Jeong-Il (Department of Microbiology, Pusan National University)
  • 투고 : 2010.06.04
  • 심사 : 2010.06.16
  • 발행 : 2010.06.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

DevSR two-component system은 Mycobacterium smegmatis의 redox sensing에 관련된 주요한 regulatory system이다. DevSR system은 DevS histidine kinase와 DevR response regulator로 구성되어 있다. 저산소 조건에서 DevS histidine kinase는 활성화되어 DevR response regulator를 인산화 시키고, 인산화된 DevR response regulator는 DevR regulon의 transcriptional activator로 작용한다. DevS의 kinase activity는 DevS의 N-terminal에 위치한 GAF domain에 존재하는 heme의 ligand-binding state에 의해 결정된다. 본 연구에서는 C-terminal kinase domain의 redox-responsive cysteine (C547)이 DevS kinase activity의 redox-dependent control과 연관이 있음을 밝혔다. 산소가 존재할 때, C547 residue 사이의 disulfide bond의 형성은 DevS kinase activity를 불활성화 시킨다. $\beta$-mercaptoethanol과 dithiothreitol과 같은 환원제를 이용하여 산화된 DevS를 환원시켰을 때, DevS kinase activity가 복원된 것이 관찰되었다. 또한, C547을 alanine으로 치환했을 때, M. smegmatis의 DevS의 sensory 기능을 부분적으로 손상되는 것이 complementation 실험을 통해 in vivo 상에서 증명되었다.

The DevSR two-component system is a major regulatory system involved in redox sensing in Mycobacterium smegmatis. The DevSR system consists of the DevS histidine kinase and its cognate DevR response regulator. When exposed to hypoxic conditions, the DevS histidine kinase is activated to phosphorylate the DevR response regulator, leading to the transcriptional activation of the DevR regulation. The ligand-binding state of the heme embedded in the N-terminal GAF domain of DevS determines the kinase activity of DevS. In this study, we demonstrated that the redox-responsive cysteine (C547) in the C-terminal kinase domain is involved in the redox-dependent control of DevS kinase activity. The formation of an intersubunit disulfide bond between the C547 residues in the presence of $O_2$ led to inactivation of DevS kinase activity. The reduction of the oxidized DevS with reductants such as $\beta$-mercaptoethanol and dithiothreitol resulted in the restoration of DevS kinase activity. It was demonstrated in vivo by complementation test that the substitution of C547 to alanine partially impaired the sensory function of DevS in M. smegmatis.

키워드

참고문헌

  1. Boon, C., R. Li, R. Qi, and T. Dick. 2001. Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model. J. Bacteriol. 183, 2672-2676. https://doi.org/10.1128/JB.183.8.2672-2676.2001
  2. Cho, H. Y., H. J. Cho, Y. M. Kim, J. I. Oh, and B. S. Kang. 2009. Structural insight into the heme-based redox sensing by DosS from Mycobacterium tuberculosis. J. Biol. Chem. 284, 13057-13067. https://doi.org/10.1074/jbc.M808905200
  3. Cunningham, A. F. and C. L. Spreadbury. 1998. Mycobacterial stationary phase induced by low oxygen tension: Cell wall thickening and localization of the 16-kilodalton alpha-crystallin homology. J. Bacteriol. 180, 801-808.
  4. Desjardin, L. E., L. G. Hayes, C. D. Sohaskey, L. G. Wayne, and K. D. Eisenach. 2001. Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis. J. Bacteriol. 183, 5311-5316. https://doi.org/10.1128/JB.183.18.5311-5316.2001
  5. Dick, T., B. H. Lee, and B. Murugasu-Oei. 1998. Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol. Lett. 163, 159-164. https://doi.org/10.1111/j.1574-6968.1998.tb13040.x
  6. Florczyk, M. A., L. A. McCue, R. F. Stack, C. R. Hauer, and K. A. McDonough. 2001. Identification and characterization of mycobacterial proteins differentially expressed understanding and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins. Infect. Immun. 69, 5777-5785. https://doi.org/10.1128/IAI.69.9.5777-5785.2001
  7. Gill, W. P., N. S. Harik, M. R. Whiddon, R. P. Liao, J. E. Mittler, and D. R. Sherman. 2009. A replication clock for Mycobacterium tuberculosis. Nat. Med. 15, 211-214. https://doi.org/10.1038/nm.1915
  8. Honaker, R. W., R. L. Leistikow, I. L. Bartek, and M. I. Voskuil. 2009. Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy. Infect. Immun. 77, 3258-3263. https://doi.org/10.1128/IAI.01449-08
  9. Howard, N. S., J. E. Gomez, C. Ko, and W. R. Bishai. 1995. Color selection with a hygromycin-resistance-based Escherichia coli-mycobacterial shuttle vector. Gene 166, 181-182. https://doi.org/10.1016/0378-1119(95)00597-X
  10. Ioanoviciu, A., E. T. Yukl, P. Moenne-Loccoz, and P. R. O. de Montellano. 2007. DevS, a heme-containing two-component oxygen sensor of Mycobacterium tuberculosis. Biochemistry 46, 4250-4260. https://doi.org/10.1021/bi602422p
  11. Jessee, J. 1986. New subcloning efficiency competent cells:>1$10^6$ transformants/${\mu}g$. Focus 8, 9.
  12. Kendall, S. L., F. Movahedzadeh, S. C. G. Rison, L. Wernisch, T. Parish, K. Duncan, J. C. Betts, and N. G. Stoker. 2004. The Mycobacterium tuberculosis dosRS two-component system is induced by multiple stresses. Tuberculosis 84, 247-255. https://doi.org/10.1016/j.tube.2003.12.007
  13. Kumar, A., J. C. Toledo, R. P. Patel, J. R. Lancaster, and A. J. C. Steyn. 2007. Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor. Proc. Natl. Acad. Sci. USA 104, 11568-11573. https://doi.org/10.1073/pnas.0705054104
  14. Lee, J. M., H. Y. Cho, H. J. Cho, I. J. Ko, S. W. Park, H. S. Baik, J. H. Oh, C. Y. Eom, Y. M. Kim, B. S. Kang, and J. I. Oh. 2008. $O_2$- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis. J. Bacteriol. 190, 6795-6804. https://doi.org/10.1128/JB.00401-08
  15. Malpica, R., B. Franco, C. Rodriguez, O. Kwon, and D. Georgellis. 2004. Identification of a quinone-sensitive redox switch in the ArcB sensor kinase. Proc. Natl. Acad. Sci. USA 101, 13318-13323. https://doi.org/10.1073/pnas.0403064101
  16. Muttucumaru, D. G. N., G. Roberts, J. Hinds, R. A. Stabler, and T. Parish. 2004. Gene expression profile of Mycobacterium tuberculosis in a non-replicating state. Tuberculosis 84, 239-246. https://doi.org/10.1016/j.tube.2003.12.006
  17. Oelmuller, U., N. Kruger, A. Steinbuchel, and C. G. Friedrich. 1990. Isolation of procaryotic RNA and detection of specific mRNA with biotinylated probes. J. Microbiol. Methods 11, 12.
  18. Park, H. D., K. M. Guinn, M. I. Harrell, R. Liao, M. I. Voskuil, M. Tompa, G. K. Schoolnik, and D. R. Sherman. 2003. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48, 833-843. https://doi.org/10.1046/j.1365-2958.2003.03474.x
  19. Podust, L. M., A. Ioanoviciu, and P. R. O. de Montellano. 2008. 2.3 angstrom X-ray structure of the heme-bound GAF domain of sensory histidine. Biochemistry 47, 12523-12531. https://doi.org/10.1021/bi8012356
  20. Roberts, D. M., R. L. P. Liao, G. Wisedchaisri, W. G. J. Hol, and D. R. Sherman. 2004. Two sensor kinases contribute to the hypoxic response of Mycobacterium tuberculosis. J. Biol. Chem. 279, 23082-23087. https://doi.org/10.1074/jbc.M401230200
  21. Saini, D. K., V. Malhotra, D. Dey, N. Pant, T. K. Das, and J. S. Tyagi. 2004. DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR. Microbiology 150, 865-875. https://doi.org/10.1099/mic.0.26218-0
  22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  23. Sardiwal, S., S. L. Kendall, F. Movahedzadeh, S. C. G. Rison, N. G. Stoker, and S. Djordjevic. 2005. A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem. J. Mol. Biol. 353, 929-936. https://doi.org/10.1016/j.jmb.2005.09.011
  24. Sherman, D. R., M. Voskuil, D. Schnappinger, R. L. Liao, M. I. Harrell, and G. K. Schoolnik. 2001. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin. Proc. Natl. Acad. Sci. USA 98, 7534-7539. https://doi.org/10.1073/pnas.121172498
  25. Snapper, S. B., R. E. Melton, S. Mustafa, T. Kieser, and W. R. Jacobs. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911-1919. https://doi.org/10.1111/j.1365-2958.1990.tb02040.x
  26. Sousa, E. H. S., J. R. Tuckerman, G. Gonzalez, and M. A. Gilles-Gonzalez. 2007. DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis. Protein Sci. 16, 1708-1719. https://doi.org/10.1110/ps.072897707
  27. Swem, L. R., B. J. Kraft, D. L. Swem, A. T. Setterdahl, S. Masuda, D. B. Knaff, J. M. Zaleski, and C. E. Bauer. 2003. Signal transduction by the global regulator RegB is mediated by a redox-active cysteine. EMBO J. 22, 4699-4708. https://doi.org/10.1093/emboj/cdg461
  28. Tabor, S. and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/ promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074-1078. https://doi.org/10.1073/pnas.82.4.1074
  29. Voskuil, M. I., K. C. Visconti, and G. K. Schoolnik. 2004. Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis 84, 218-227. https://doi.org/10.1016/j.tube.2004.02.003
  30. Wayne, L. G. and C. D. Sohaskey. 2001. Nonreplicating persistence of Mycobacterium tuberculosis. Annu. Rev. Microbiol. 55, 139-163. https://doi.org/10.1146/annurev.micro.55.1.139
  31. Yuan, Y., D. D. Crane, R. M. Simpson, Y. Q. Zhu, M. J. Hickey, D. R. Sherman, and C. E. Barry. 1998. The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc. Natl. Acad. Sci. USA 95, 9578-9583. https://doi.org/10.1073/pnas.95.16.9578