DOI QR코드

DOI QR Code

R. sphaeroides 에서의 orf282 유전자의 분석과 이들의 기능

Analysis of the orf 282 Gene and Its Function in Rhodobacter sphaeroide 2.4.1

  • 손명화 (부산대학교 미생물학과) ;
  • 이상준 (부산대학교 미생물학과)
  • Son, Myung-Hwa (Department of Microbiology, Pusan National University) ;
  • Lee, Sang-Joon (Department of Microbiology, Pusan National University)
  • 투고 : 2012.06.13
  • 심사 : 2012.07.11
  • 발행 : 2012.08.30

초록

Rodobacter sphaeroides에서 orf282 유전자는 cbb3 terminal oxidase를 암호화하는 ccoNOQP 오페론과 혐기적 활성자인 FnrL을 암호화하는 fnrL 유전자 사이에 있으며, 아직은 기능이 잘 알려지지 않았다. orf282 유전자의 기능을 알기 위해 우리는 orf282의 일부를 삭제함으로써 유전자를 붕괴시켜 orf282-minus mutant를 제조하였다. 두개의 FnrL 결합 부위가 orf282의 upstream에 존재한다는 것이 밝혀져 있으며, orf282 유전자가 FnrL에 의해 양성적으로 조절된다는 것이 증명되었다. orf282 유전자는 B875와 B800-850 spectral complexes의 형성과 관련이 없다. orf282 mutant에서의 cbb3 oxidase 활성을 wild type와 비교해보면 orf282 유전자가 ccoNOQP 오페론의 조절과 cbb3 cytochrome c oxidase의 생합성과 무관하다는 것을 알 수 있다. orf282 mutant의 구조 유전자인 nifH와 조절유전자인 nifA의 프로모터 활성이 증가한 것은 orf282 유전자 산물이 nifH와 nifA의 발현에서 음성적 effector로 작용한다는 것을 시사한다.

The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding $cbb_3$ terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The $cbb_3$ oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

키워드

참고문헌

  1. Aravind, L., Anantharaman, V. and Koonin, E. V. 2002. Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA world. Proteins 48, 1-14. https://doi.org/10.1002/prot.10064
  2. Cohen-Bazire, G., Sistrom, W. R. and Stanier, R. Y. 1957. Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Physiol. 49, 25-68. https://doi.org/10.1002/jcp.1030490104
  3. Davis, J., Donohue, T. J. and Kaplan, S. 1988. Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides. J. Bacteriol. 170, 320-329.
  4. Diez, A. A., Gustavsson, N. and Nyström, T. 2000. The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway. Mol. Microbiol. 36, 1494-1503.
  5. Diez, A. A., Farewell, T., Nannmark, U. and Nyström, T. 1997. A mutation in the ftsK gene of Escherichia coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J. Bacteriol. 179, 5878-5883.
  6. de Gier, J. W., Schepper, M., Reijnders, W. N., van Dyck, S. J., Slotboom, D. J., Warne, A., Saraste, M., Krab, K., Finel, M., Stouthamer, A. H., van Spanning, R. J. and van der Oost, J. 1996. Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design. Mol. Microbiol. 20, 1247-1260. https://doi.org/10.1111/j.1365-2958.1996.tb02644.x
  7. Farewell, A., Diez, A. A., DiRusso, C. C. and Nyström, T. 1996. Role of the Escherichia coli FadR regulator in stasis survival and growth phase-dependent expression of the uspA, fad, and fab genes. J. Bacteriol. 178, 6443-6450.
  8. Fostner-Hartnett, D. and Kranz, R. G. 1992. Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus: activation requires ntrC but not rpoN. Mol. Microbiol. 6, 1049-1060. https://doi.org/10.1111/j.1365-2958.1992.tb02170.x
  9. Freestone, P., Nystrom, T., Trinei, M. and Norris, V. 1997. The universal stress protein, UspA, of Escherichia coli is phosphorylated in response to stasis. J. Mol. Biol. 274, 318-324. https://doi.org/10.1006/jmbi.1997.1397
  10. Gustavsson, N., Diez, A. A. and Nyström, T. 2002. The universal stress protein paralogues of Escherichia coli are co-ordinately regulated and co-operate in the defence against DNA damage. Mol. Microbiol. 43, 107-117. https://doi.org/10.1046/j.1365-2958.2002.02720.x
  11. Halbleib, C. M. and Ludden, P. W. 2000. Regulation of biological nitrogen fixation. J. Nutr. 130, 1081-1084.
  12. Hubner, P., Willison, J. C., Vignais, P. M. and Bickle, T. A. 1991. Expression of regulatory nif genes in Rhodobacter capsulatus. J. Bacteriol. 173, 2993-2999.
  13. Jessee, J. 1986. New subcloning efficiency competent cells: >$1{\times}10^6$ transformants/${\mu}g$. Focus 8, 9.
  14. Joshi, H. M. and Tabita, F. R. 1996. A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation. Proc. Natl. Acad. Sci. USA 93, 14515-14520. https://doi.org/10.1073/pnas.93.25.14515
  15. Koch, H.-G., Hwang, O. and Daldal, F. 1998. Isolation and characterization of Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity. J. Bacteriol. 180, 969-978.
  16. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. and Peterson, K. M. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176. https://doi.org/10.1016/0378-1119(95)00584-1
  17. Kranz, R. G. and Foster-Hartnett, D. 1990. Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen and nitrogen-responsive factors. Mol. Microbiol. 4, 1793-1800. https://doi.org/10.1111/j.1365-2958.1990.tb02027.x
  18. Kvint, K., Nachin, L., Diez, A. and Nyström, T. 2003. The bacterial universal stress protein: function and regulation. Curr. Opin. Microbiol. 6, 140-145. https://doi.org/10.1016/S1369-5274(03)00025-0
  19. Kvint, K., Hosbond, C., Farewell, A., Nybroe, O. and Nystrom, T. 2000. Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor. Mol. Microbiol. 35, 435-443. https://doi.org/10.1046/j.1365-2958.2000.01714.x
  20. Lee, J. K. and Kaplan, S. 1995. Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides. Analysis of the cis-acting downstream regulatory sequence. J. Biol. Chem. 270, 20453-20458. https://doi.org/10.1074/jbc.270.35.20453
  21. Lenz, O., Schwartz, E., Dernedde, J., Eitinger, M. and Friedrich, B. 1994. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation. J. Bacteriol. 176, 4385-4393.
  22. Madigan, M. T. and Gest, H. 1979. Growth of the photosynthetic bacterium Rhodopseudomonas capsulatus chemoautotrophically in darkness with $H_2$ as the energy source. J. Bacteriol. 137, 524-530.
  23. Makarova, K. S., Aravind, L. Galperin, M. Y., Grishin, N. V., Tatusov, R. L., Wolf, Y. I. and Koonin, E. V. 1999. Comparative genomics of the Archaea (Euryarchaeota): evolution of conserved protein families, the stable core, and the variable shell. Genome Res. 9, 608-628.
  24. Meinhardt, S. W., Kiley, P. J., Kaplan, S., Crofts, A. R. and Harayama, S. 1985. Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light-harvesting complexes to the reaction center. Arch. Biochem. Biophys. 236, 130-139. https://doi.org/10.1016/0003-9861(85)90612-5
  25. Mushegian, A. R. and Koonin, E. V. 1996. Sequence analysis of Eukaryotic developmental proteins: ancient and novel domains. Genetics 144, 817-828.
  26. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
  27. Nystrom, T. and Gustavsson, N. 1998. Maintenance energy requirement: what is required for stasis survival of Escherichia coli? Biochim. Biophys. Acta. 1365, 225-231. https://doi.org/10.1016/S0005-2728(98)00072-3
  28. Nystrom, T. and Neidhardt, F. C. 1994. Expression and role of the universal stress protein. UspA, of Escherichia coli during growth arrest. Mol. Microbiol. 11, 537-544. https://doi.org/10.1111/j.1365-2958.1994.tb00334.x
  29. Nystrom, T. and Neidhardt, F. C. 1992. Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Eschericha coli. Mol. Microbiol. 6, 3187-3198. https://doi.org/10.1111/j.1365-2958.1992.tb01774.x
  30. Oelze, J. and Klein, G. 1996. Control of nitrogen fixation by oxygen in purple nonsulfur bacteria. Arch. Microbiol. 165, 219-225. https://doi.org/10.1007/s002030050319
  31. Oh, J.-I. and Kaplan, S. 1999. The cbb3 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
  32. Persson, O., Valadi, A., Nyström, T. and Farewell, A. 2007. Metabolic control of the Escherichia Coli universal stress protein response through fructose-6-phosphate. Mol. Microbiol. 65, 968-978. https://doi.org/10.1111/j.1365-2958.2007.05838.x
  33. Preisig, O., Anthamatten, D. and Hennecke, H. 1993. Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. Proc. Natl. Acad. Sci. USA 90, 3309-3313. https://doi.org/10.1073/pnas.90.8.3309
  34. Revers, L. F., Passaglia, L. M. P., Marchal, K., Frazzon, J., Blaha, C. G. Vanderleyden, J. and Schrank, I. S. 2000. Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N2 fixation: the effect of ORF280 on nifH expression. FEMS. Microbiol. Lett. 183, 23-29. https://doi.org/10.1111/j.1574-6968.2000.tb08928.x
  35. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  36. Siegele, D. A. 2005. Universal stress proteins in Escherichia coli. J. Bacteriol. 187, 6253-6254. https://doi.org/10.1128/JB.187.18.6253-6254.2005
  37. Simon, R., Priefer, U. and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1, 784-791. https://doi.org/10.1038/nbt1183-784
  38. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  39. van Niel, C. B. 1944. The culture, general physiology, morphology, and classification of the non-sulfur purple and brown bacteria. Bacteriol. Rev. 8, 1-118.
  40. Zeilstra-Ryalls, J. H. and Kaplan, S. 2004. Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm. Cell 61, 417-436.
  41. Zeilstra-Ryalls, J. H. and Kaplan, S. 1995. Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene. J. Bacteriol. 177, 6422-6431.
  42. Zufferey, R., Preisig, O., Hennecke, H. and Thony-Meyer, L. 1996. Assembly and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum. J. Biol. Chem. 271, 9114-9119. https://doi.org/10.1074/jbc.271.15.9114

피인용 문헌

  1. The Role of NifA and PrrA on the Expression of nif Gene in Rhodobacter sphaeroides vol.21, pp.9, 2012, https://doi.org/10.5322/JES.2012.21.9.1139