Cloning and Molecular Characterization of groESL Heat-Shock Operon in Methylotrophic Bacterium Methylovorus Sp. Strain SS1 DSM 11726

  • Eom, Chi-Yong (Department of Biology, Yonsei University) ;
  • Kim, Eung-Bin (Department of Biology, Yonsei University) ;
  • Ro, Young-Tae (Laboratory of Biochemistry, College of Medicine, Konkuk University) ;
  • Kim, Si-Wouk (Department of Environmental Engineering, Chosun University) ;
  • Kim, Young-Min (Department of Biology, Yonsei University)
  • Published : 2005.11.30


The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli ${\sigma}^{32}$-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10min after increasing the temperature from 30 to $42^{\circ}C$. The groESL operon was also induced by hydrogen peroxide or salt shock.


  1. Ang, D., Liberek, K., Skowyra, D., Zylicz, M. and Georgopoulos, C. (1991) Biological role and regulation of the universally conserved heat shock proteins. J. Biol. Chem. 266, 24233- 24236
  2. Avedissian, M., and Lopes Gomes, S. (1996) Expression of the groESL operon is cell-cycle controlled in Caulobacter crescentus. Mol. Microbiol. 19, 79-89
  3. Babst, M., Hennecke, H. and Fischer, H. M. (1996) Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum. Mol. Microbiol. 19, 827-839
  4. Becker, J. and Craig, E. A. (1994) Heat-shock proteins as molecular chaperones. Eur. J. Biochem. 219, 11-23
  5. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
  6. Bratina, B. J., Brusseau, G. A. and Hanson, R. S. (1992) Use of 16S rRNA analysis to investigate phylogeny of methylotrophic bacteria. Intl. J. Syst. Bacteriol. 42, 645-648
  7. Bulygina, E. S., Galchenko, V. F., Govorukhina, N. I., Netrusov, A. I., Nikitin, D. I., Trotsenko, Y. A. and Chumakov, K. M. (1990) Taxomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing. J. Gen. Microbiol. 136, 441-446
  8. Chain, P., Lamerdin, J., Larimer, F., Regala, W., Lao, V., Land, M., Hauser, L., Hooper, A., Klotz, M., Norton, J., Sayavedra- Soto, L., Arciero, D., Hommes, N., Whittaker, M. and Arp, D. (2003) Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 185, 2759-2773
  9. Cowing, D. W., Bardwell, J. C., Craig, E. A., Woolford, C., Hendrix, R. W. and Gross, C. A. (1985) Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 82, 2679-2683
  10. Craig, E. A., Gambill, B. D. and Nelson, R. J. (1993) Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol. Rev. 57, 402-414
  11. Ellis, R. J. and van der Vies, S. M. (1991) Molecular chaperones. Annu. Rev. Biochem. 60, 321-347
  12. Eom, C. Y., Park, S. T., Kim, E., Ro, Y. T., Kim, S. W. and Kim, Y. M. (2002) Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726. Mol. Cells 14, 245-254
  13. Ewalt, K. L., Hendrick, J. P., Houry, W. A. and Hartl, F. U. (1997) In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell 90, 491-500
  14. Fayet, O., Ziegelhoffer, T. and Georgopoulos, C. (1989) The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171, 1379-1385
  15. Furuki, M., Tanaka, N., Hiyama, T. and Nakamoto, H. (1996) Cloning, characterization and functional analysis of groEL-like gene from thermophilic cyanobacterium Synechococcus vulcanus, which does not form an operon with groES. Biochim. Biophys. Acta 1294, 106-110
  16. Georgopoulos, C. and Welch, W. J. (1993) Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell. Biol. 9, 601-634
  17. Goldberg, J. B. and Ohman, D. E. (1984) Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J. Bacteriol. 158, 1115-1121
  18. Hartl, F. U. (1996) Molecular chaperones in cellular protein folding. Nature 381, 571-579
  19. Hartl, F. U. and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-1858
  20. Hecker, M., Schumann, W. and Volker, U. (1996) Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19, 417-428
  21. Hendrick, J. P. and Hartl, F. U. (1993) Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62, 349- 384
  22. Horwich, A. L., Low, K. B., Fenton, W. A., Hirshfield, I. N. and Furtak, K. (1993) Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 74, 909-917
  23. Houry, W. A. (2001) Chaperone-assisted protein folding in the cell cytoplasm. Curr. Protein Pept. Sci. 2, 227-244
  24. Iizumi, T. and Nakamura, K. (1997) Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene. Appl. Environ.Microbiol. 63, 1777-1784
  25. Kim, Y. M. and Hegeman, G. D. (1981) Purification and some properties of carbon monoxide dehydrogenase from Pseudomonas carboxydohydrogena. J. Bacteriol. 148, 904-911
  26. Lee, W. T. Terlesky, K. C. and Tabita, F. R. (1997) Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon. J. Bacteriol. 179, 487-495
  27. Lidstrom, M. E. and Stirling, D. I. (1990) Methylotrophs: genetics and commercial applications. Annu. Rev. Microbiol. 44, 27-58
  28. Lindquist, S. and Craig, E. A. (1988) The heat-shock proteins. Annu. Rev. Genet. 22, 631-677
  29. McLennan, N. F., Girshovich, A. S., Lissin, N. M., Charters, Y. and Masters, M. (1993) The strongly conserved carboxylterminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable. Mol. Microbiol. 7, 49-58
  30. Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F. X. and Schumann, W. (1997) The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J. 16, 4579-4590
  31. Park, J. H., Kim, S. W., Kim, E., Ro, Y. T. and Kim, Y. M. (2001) Stress-shock response of a methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726. J. Microbiol. 37, 162-167
  32. Rajaram, H., Ballal, A. D., Apte, S. K., Wiegert, T. and Schumann, W. (2001) Cloning and characterization of the major groESL operon from a nitrogen-fixing cyanobacterium Anabaena sp. strain L-31. Biochim. Biophys. Acta 1519, 143- 146
  33. Roberts, R. C., Toochinda, C., Avedissian, M., Baldini, R. L., Gomes, S. L. and Shapiro, L. (1996) Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE. J. Bacteriol. 178, 1829-1841
  34. Rusanganwa, E. and Gupta, R. S. (1993) Cloning and characterization of multiple groEL chaperonin-encoding genes in Rhizobium meliloti. Gene 126, 67-75
  35. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, USA
  36. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467
  37. Segal, G. and Ron, E. Z. (1993) Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J. Bacteriol. 175, 3083-3088
  38. Segal, G. and Ron, E. Z. (1996) Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat. J. Bacteriol. 178, 3634- 3640
  39. Segal, G. and Ron, E. Z. (1998) Regulation of heat-shock response in bacteria. Ann. New York Acad. Sci. 851, 147-151
  40. Seo, S. A. and Kim, Y. M. (1993) Isolation and characterization of a restricted facultatively methylotrophic bacterium Methylovorus sp. strain SS1. Kor. J. Microbiol. 31, 179-183
  41. Shine, J. and Dalgarno, L. (1974) The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342-1346
  42. Tettelin, H., Saunders, N. J., Heidelberg, J., Jeffries, A. C., Nelson, K. E., Eisen, J. A., Ketchum, K. A., Hood, D. W., Peden, J. F., Dodson, R. J., Nelson, W. C., Gwinn, M. L., DeBoy, R., Peterson, J. D., Hickey, E. K., Haft, D. H., Salzberg, S. L., White, O., Fleischmann, R. D., Dougherty, B. A., Mason, T., Ciecko, A., Parksey, D. S., Blair, E., Cittone, H., Clark, E. B., Cotton, M. D., Utterback, T. R., Khouri, H., Qin, H., Vamathevan, J., Gill, J., Scarlato, V., Masignani, V., Pizza, M., Grandi, G., Sun, L., Smith, H. D., Fraser, C. M., Moxon, E. R., Rappuoli, R. and Venter, J. C. (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809-1815
  43. Tilly, K. and Georgopoulos, C. (1982) Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo. J. Bacteriol. 149, 1082-1088
  44. Tilly, K., Murialdo, H. and Georgopoulos, C. (1981) Identification of a second Escherichia coli groE gene whose product is necessary for bacteriophage morphogenesis. Proc. Natl. Acad. Sci. USA 78, 1629-1633
  45. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354
  46. Yura, T., Nagai, H. and Mori, H. (1993) Regulation of the heatshock response in bacteria. Annu. Rev. Microbiol. 47, 321-350
  47. Zhou, Y. N., Kusukawa, N., Erickson, J. W., Gross, C. A. and Yura, T. (1988) Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32. J. Bacteriol. 170, 3640-3649
  48. Zuber, U. and Schumann, W. (1994) CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176, 1359-1363

Cited by

  1. Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice vol.4, pp.1, 2011,