Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Identification of Amino Acids Involved in the Sensory Function of the PrrB Histidine Kinase by Site-directed Mutagenesis

Site-directed mutagenesis에 의한 PrrB histidine kinase의 신호인지 기능에 관련된 아미노산의 발굴

  • Kim Yong-Jin (Department of Microbiology, Pusan National University) ;
  • Ko In-Jeong (Korea Science Academy) ;
  • Oh Jeong-Il (Department of Microbiology, Pusan National University)
  • 김용진 (부산대학교 자연과학대학 미생물학과) ;
  • 고인정 (한국과학영재학교) ;
  • 오정일 (부산대학교 자연과학대학 미생물학과)
  • Published : 2006.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

The PrrBA two-component system is one of the major regulatory systems that control expression of photosynthesis genes in response to changes in oxygen tension in the anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The system consists of the PrrB histidine kinase and the PrrA response regulator. The N-terminal transmembrane domain of PrrB serves as a signal-sensing domain and comprises six transmembrane helices forming three periplasmic loops and two cytoplasmic loops. The $3^{rd}$ and $4^{th}$ transmembrane helices and the $2^{nd}$ periplasmic loop were suggested to play a crucial role in redox-sensory function. In this study we demonstrated that mutations of Asp-90, Gln-93, Leu-94, Leu-98, and Asn-106 in the $2^{nd}$ periplasmic loop and its neighboring region led to severe defects in PrrB sensory function, indicating that these amino acids might be related to the redox-sensing function of PrrB. The mutant forms (D90E, D90N, and D90A) of PrrB were heterologously overexpressed in Escherichia coli, purified by means of affinity chromatography and their autokinase activities were comparatively assessed. The D90N form of PrrB was shown to possess higher autokinase activity than the wild-type form of PrrB, whereas the D90E form of PrrB displayed lower autokinase activity than the wild-type form of PrrB. The D90A mutation led to the loss of PrrB autokinase activity.

광합성세균인 Rhodobacter sphaeroides의 PrrBA two-component system은 산소분압의 변화에 따라 광합성 유전자의 발현을 조절하는 주요한 조절계 중 하나이다. PrrBA two-component system은 PrrB histidine kinase와 PrrA response regulator로 구성되어 있는데, PrrB의 N-말단 transmembrane 도메인은 신호인지 도메인으로서, 여섯 개의 transmembrane helix가 세 개의 periplasmic loop와 두 개의 cytoplasmic loop을 형성하고 있다. 그 중 세 번째, 네 번째 transmembrane helix와 두 번째 periplasmic loop가 산화/환원 인지 기능에 있어 중요한 역할을 할 것이라고 제안되었다. 본 연구에서는, 두 번째 periplasmic loop와 그 인접 부위에서의 돌연변이 (Asp-90, Gln-93, Leu-94, Leu-98, Asn-106)에 의해 PrrB의 인지 기능에 있어 심각한 결함이 생기는 것을 증명하였고, 이는 이 아미노산들이 PrrB의 산화/환원 인지 기능에 연관되어 있을 수 있다는 것을 보여준다. PrrB의 돌연변이 형태 (D90E, D90N, D90A)가 대장균에서 과발현되어서 affinity chromatography에 의해 정제되었고, 정제된 단백질의 자가인산화 반응이 측정되었다. PrrB D90N 변이형태는 PrrB wild-type보다 높은 자가인산화 활성을 가지는 반면에, PrrB D90E 변이형태는 PrrB wild-type보다 낮은 자가인산화 활성을 나타내었다. 그리고 D90A 변이형태는 PrrB의 자가인산화 활성이 매우 약화되었다.

Keywords

References

  1. Choudhary, M., and S. Kaplan. 2000. DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1. Nucleic Acids Res. 28, 862-867 https://doi.org/10.1093/nar/28.4.862
  2. Comolli, J.C., A. J. Carl, C. Hall, and T. Donohue. 2002. Transcriptional activation of the Rhodobacter sphaeroides cytochrome $c_2$ gene P2 promoter by the response regulator PrrA. J. Bacteriol. 184, 390-399 https://doi.org/10.1128/JB.184.2.390-399.2002
  3. Davis, J., T. J. Donohue, and S. Kaplan. 1988. Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides. J. Bacteriol. 170, 320-329 https://doi.org/10.1128/jb.170.1.320-329.1988
  4. Eraso, J. M., and S. Kaplan. 1994. prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides. J. Bacteriol. 176, 32-43 https://doi.org/10.1128/jb.176.1.32-43.1994
  5. Eraso, J. M., and S. Kaplan. 1995. Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase. J. Bacteriol. 177, 2695-2706 https://doi.org/10.1128/jb.177.10.2695-2706.1995
  6. Eraso, J. M., and S. Kaplan. 1996. Complex regulatory activities associated with the histidine kinase PrrB in expression of photosynthesis genes in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 178, 7037-7046 https://doi.org/10.1128/jb.178.24.7037-7046.1996
  7. Gomelsky, M., and S. Kaplan. 1995. appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 177, 4609-4618 https://doi.org/10.1128/jb.177.16.4609-4618.1995
  8. Gomelsky, M., and S. Kaplan. 1995. Genetic evidence that PpsR from Rhodobacter sphaeroides 2.4.1 functions as a repressor of puc and bchF expression. J. Bacteriol. 177, 1634-1637 https://doi.org/10.1128/jb.177.6.1634-1637.1995
  9. Kaplan, S., J. M. Eraso, and J. H. Roh. 2005. Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1. Biochem. Soc. Trans. 33, 51-55 https://doi.org/10.1042/BST0330051
  10. Keen, N. T., S. Tamaki, D. Kobayashi, and D. Trollinger. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191-197 https://doi.org/10.1016/0378-1119(88)90117-5
  11. Kiley, P. J., and S. Kaplan. 1988. Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol. Rev. 52, 50-69
  12. Kobe, B., and J. Deisenhofer. 1994. The leucine-rich repeat: a versatile binding motif. Trends Biochem. Sci. 19, 415-421 https://doi.org/10.1016/0968-0004(94)90090-6
  13. O'Gara, J.P., J. M. Eraso, and S. Kaplan. 1998. A redox- responsive pathway for aerobic regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J Bacteriol. 180, 4044-4050
  14. O'Gara, J. P., and S. Kaplan. 1997. Evidence for the role of redox carriers in photosynthesis gene expression and carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 179, 1951-1961 https://doi.org/10.1128/jb.179.6.1951-1961.1997
  15. 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, 73-84 https://doi.org/10.1016/0167-7012(90)90050-G
  16. Oh, J. I., J. M. Eraso, and S. Kaplan. 2000. Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 182, 3081-3087 https://doi.org/10.1128/JB.182.11.3081-3087.2000
  17. Oh, J. I., and S. Kaplan. 1999. The $cbb_3$ terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation. Biochemistry 38, 2688-2696 https://doi.org/10.1021/bi9825100
  18. Oh, J. I., and S. Kaplan. 2000. Redox signaling: globalization of gene expression. EMBO J. 19, 4237-4247 https://doi.org/10.1093/emboj/19.16.4237
  19. Oh, J. I., and S. Kaplan. 2001. Generalized approach to the regulation and integration of gene expression. Mol. Microbiol. 39, 1116-1123 https://doi.org/10.1111/j.1365-2958.2001.02299.x
  20. Oh, J. I., I. J. Ko, and S. Kaplan. 2001. The default state of the membrane-localized histidine kinase PrrB of Rhodobacter sphaeroides 2.4.1 is in the kinase-positive mode. J. Bacteriol. 183, 6807-6814 https://doi.org/10.1128/JB.183.23.6807-6814.2001
  21. Oh, J. I., I. J. Ko, and S. Kaplan. 2004. Reconstitution of the Rhodobacter sphaeroides $cbb_3-PrrBA$ signal transduction pathway in vitro. Biochemistry 43, 7915-7923 https://doi.org/10.1021/bi0496440
  22. Ouchane, S., and S. Kaplan. 1999. Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 274, 17290-17296 https://doi.org/10.1074/jbc.274.24.17290
  23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Swem, L. R., X. Gong, C. A. Yu, and C. E. Bauer. 2006. Identification of a ubiquinone binding site that affects autophosphorylation of the sensor kinase RegB. J. Biol. Chem. 281, 6768-6775 https://doi.org/10.1074/jbc.M509687200
  25. Zeilstra-Ryalls, J. H., and S. Kaplan. 1996. Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state. J. Bacteriol. 178, 985-993 https://doi.org/10.1128/jb.178.4.985-993.1996
  26. Zeilstra-Ryalls, J. H., and S. Kaplan. 1998. Role of the fnrL gene in photosystem gene expression and photosynthetic growth of Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 180, 1496-1503