Methylovorus sp. Strain SS1 DSM11726으로부터 rpoH 유전자의 클로닝과 염기서열 분석

Cloning and Nucleotide Sequence Analysis of the rpoH Gene from Methylovorus sp. Strain SS1 DSM11726

  • 엄치용 (한국기초과학지원연구원 서울센터 메타볼롬분석연구팀) ;
  • 송승은 (한국기초과학지원연구원 서울센터 메타볼롬분석연구팀) ;
  • 박미화 (한국기초과학지원연구원 서울센터 메타볼롬분석연구팀) ;
  • 김영민 (연세대학교 이과대학 생물학과)
  • Eom, Chi-Yong (Metabolome Analysis Team, Korea Basic Science Institute (KBSI)) ;
  • Song, Seung-Eun (Metabolome Analysis Team, Korea Basic Science Institute (KBSI)) ;
  • Park, Mi-Hwa (Metabolome Analysis Team, Korea Basic Science Institute (KBSI)) ;
  • Kim, Young-Min (Department of Biology, College of Science, Yonsei University)
  • 발행 : 2007.09.28

초록

열충격 시그마인자를 코딩하는 유전자 rpoH가 결여된 돌연변이체 대장균(Escherichia coli satrain A7448)을, 메탄올 자화세균인 Methylovorus sp. strain SS1 DSM11726의 phagemid library로 형질전환 시켜서 $30^{\circ}C$에서 성장하는 Escherichia coli strain A7448 로부터 Methylovorus sp. strain SS1 DSM11726의 rpoH 유전자를 클로닝하고 그 염기서열을 분석하였다. 1,793-bp 염기서열 분석 결과 Methylovorus sp. strain SS1 DSM11726의 RpoH는 284개의 아미노산으로 이루어져 있었으며 예상된 분자량은 32,006, p1값은 5.79로 나타났으며, 동일계열의 ${\beta}$-proteobacteria에 속하는 세균들의 RpoH와 높은 상동성을 보여주었다. Methylovorus sp. strain SS1 DSM11726의 RpoH는 대장균의 RpoH의 기능을 대신할 수 있음을 보여주었다. 열충격 후 RpoH양은 15분까지 지속적으로 증가하다 20분 뒤 양이 감소하는 양상을 나타내었다. 이는 Methylovorus sp. strain SS1 DSM11726의 RpoH 단백질 역시 열에 의해 유도됨을 말해 준다.

Using complementation of RpoH deficient E. coli strain A7448, the rpoH gene encoding heat shock sigma factor 32 (${\sigma}^{32}$) from Methylovorus sp. strain SS1 DSM11726 was cloned and sequenced. Sequence analysis of a stretch of 1,796-bp revealed existence of an open reading frame encoding a polypeptide of 284 amino acid (32,006 dalton). Deduced amino acid sequence of the Methylovorus sp. strain SS1 RpoH showed that 59.6%, 39.1% and 51.4% identities with those of Nitrosomonas europaea (${\beta}$-proteobacteria), Agrobacterium tumefaciens ($\alpha$-proteobacteria) and E. coli (${\gamma}$-proteobacteria). The expression level of the functional ortholog of RpoH of Methylovorus sp. strain SS1 was increased transiently after heat induction, further indicating that it functions as a heat shock sigma factor.

키워드

참고문헌

  1. Benvenisti, L., S. Koby, A. Rutman, H. Giladi, T. Yura, and A. B. Oppenheim. 1995. Cloning and primary sequence of the rpoH gene from Pseudomonas aeruginosa. Gene 155: 73-76 https://doi.org/10.1016/0378-1119(94)00829-H
  2. Bibb, M. J., P. R. Findlay, and M. W. Johnson. 1984. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene 30: 157-16 https://doi.org/10.1016/0378-1119(84)90116-1
  3. Bukau, B. 1993. Regulation of the Escherichia coli heatshock response. Mol. Microbiol. 9: 671-680 https://doi.org/10.1111/j.1365-2958.1993.tb01727.x
  4. Dower, W. J., J. F. Miller, and C. W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: 6127-6145 https://doi.org/10.1093/nar/16.13.6127
  5. Eom, C. Y, S. T. Park, E. Kim, Y. T. Ro, S. W. Kim and Y M. Kim. 2002. Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp. strain SSI DSM 11726. Mol. Cells. 14: 245-254
  6. Eom, C. Y, E. Kim, Y. T. Ro, S. W. Kim, and Y M. Kim. 2005. Cloning and molecular characterization of groESL heat-shock operon in a methylotrophic bacterium Methylovarus sp. strain SS I DSM 11726. J. Biochem. Mol. Biol. 38: 695-702
  7. Gamer, J., G Multhaup, T. Tomoyasu, J. S. McCarty, S. Rudiger, H. J. Schonfeld, C. Schirra, H. Bujard, and 8. Bukau. 1996. A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor ${\sigma}^{32}$. EMBO J. 15: 607-617
  8. Georgopoulos, C., K. Liberek, M. ZyIicz, and D. Ang. 1994. Properties of heat shock proteins of Escherichia coli. and the autoregulation of the heat shock response. In: Morimoto, R. I., A. Tissires and C. Georgopoulos (eds): The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, pp 209-249
  9. Goldberg, J. B., and D. E. Ohman. 1984. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J. Bacteriol. 158:1115-1121
  10. Gross, C. A., D. B. Straus, J. W. Erickson, and T. Yura. 1990. The function and regulation of heat shock proteins in Escherichia coli. In: Morimoto, R. I, A. Tissires and C. Georgopoulos (eds): Stress proteins in biology and medicine, Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, pp 167-189
  11. Grossman, A. D., D. B. Strauss, W. A. Walter, and C. A. Gross. 1987. ${\sigma}^{32}$ synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev. 1: 179-184 https://doi.org/10.1101/gad.1.2.179
  12. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580 https://doi.org/10.1016/S0022-2836(83)80284-8
  13. LaemmIi, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  14. Lidstrom, M. E. and Stirling, D, I. 1990. Methylotrophs: genetics and commercial applications. Annu. Rev. Microbiol. 44: 27-58 https://doi.org/10.1146/annurev.mi.44.100190.000331
  15. Morimoto, R. I., A. Tissires, and C. Georgopoulos. 1994. The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor Laboratory Press. New York. Cold Spring Harbor. pp 610
  16. Nagai, H., H. Yuzawa, and T. Yura. 1991. Interplay of two cis-acting mRNA regions in translational control of ${\sigma}^{32}$ synthesis during the heat shock response of Escherichia coli. Proc. Natl. Acad. Sci. USA 88: 10515- 10519
  17. Nagai, H., H. Yuzawa, M. Kanemori, and T. Yura. 1994. A distinct segment of the ${\sigma}^{32}$ polypeptide is involved in DnaKmediated negative control of the heat shock response in Escherichia coli. Proc. Natl. Acad. Sci. USA 91: 10280- 10284
  18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbour Laboratory, Cold Spring harbour. NY
  19. Schumann, W. 1996. Regulation of the heat shock response in Escherichia coli and Bacillus subtilis. J. Biosci. 21: 133-148 https://doi.org/10.1007/BF02703104
  20. Sprengart, M. L., H. P. Fatscher, and E. Fuchs. 1990. The initiation of translation in E. coli: apparent base pairing between the 16S rRNA and downstream sequences of the mRNA. Nucleic Acids Res. 18: 1719-1723 https://doi.org/10.1093/nar/18.7.1719
  21. Straus, D. B., W. A. Walter, and C. A. Gross. 1987. The heat shock response of E. coli is regulated by changes in the concentration of ${\sigma}^{32}$. Nature 329: 348-351 https://doi.org/10.1038/329348a0
  22. Straus, D. B., W. A. Walter, and C. A. Gross. 1989. The activity of ${\sigma}^{32}$ is reduced under conditions of excess heat shock protein production in Escherichia coli. Genes Dev. 3: 2003-2010 https://doi.org/10.1101/gad.3.12a.2003
  23. Tilly, K., J. Spence, and C. Georgopoulos. 1989. Modulation of stability of the Escherichia coli heat shock regulatory factor ${\sigma}^{32}$. J Bacteriol. 171: 1585-1589 https://doi.org/10.1128/jb.171.3.1585-1589.1989
  24. Wsten, M. M. 1998. Eubacterial sigma-factors. FEMS Microbiol. Rev. 22: 127-150 https://doi.org/10.1016/S0168-6445(98)00011-4
  25. Yamarnori, T., and T. Yura. 1982. Genetic control of heatshock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 79: 860-864
  26. Yura, T., H. Nagai, and H. Mori. 1993. Regulation of the heat-shock response in bacteria. Annu. Rev. Microbiol. 47: 321-350 https://doi.org/10.1146/annurev.mi.47.100193.001541
  27. Yuzawa, H., H. Nagai, H. Mori, and T. Yura. 1993. Heat induction of sigma 32 synthesis mediated by mRNA secondary structure: a primary step of the heat shock response in Escherichia coli. Nucleic Acids Res. 21: 5449-5455 https://doi.org/10.1093/nar/21.23.5449
  28. Zhou, Y. -N., N. Kusukawa, J. W. Erickson, C. A. Gross, and T. Yura. 1988. Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor ${\sigma}^{32}$. J. Bacteriol. 170: 3640-3649 https://doi.org/10.1128/jb.170.8.3640-3649.1988