Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Nov;176(22):6965–6973. doi: 10.1128/jb.176.22.6965-6973.1994

Isolation and characterization of point mutations in the Escherichia coli grpE heat shock gene.

B Wu 1, D Ang 1, M Snavely 1, C Georgopoulos 1
PMCID: PMC197068  PMID: 7961459

Abstract

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure-function analysis of the GrpE protein.

Full text

PDF
6965

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ang D., Georgopoulos C. The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds. J Bacteriol. 1989 May;171(5):2748–2755. doi: 10.1128/jb.171.5.2748-2755.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolliger L., Deloche O., Glick B. S., Georgopoulos C., Jenö P., Kronidou N., Horst M., Morishima N., Schatz G. A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. EMBO J. 1994 Apr 15;13(8):1998–2006. doi: 10.1002/j.1460-2075.1994.tb06469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bork P., Sander C., Valencia A., Bukau B. A module of the DnaJ heat shock proteins found in malaria parasites. Trends Biochem Sci. 1992 Apr;17(4):129–129. doi: 10.1016/0968-0004(92)90319-5. [DOI] [PubMed] [Google Scholar]
  4. Bucca G., Smith C. P., Alberti M., Seidita G., Passantino R., Puglia A. M. Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2). Gene. 1993 Aug 16;130(1):141–144. doi: 10.1016/0378-1119(93)90358-a. [DOI] [PubMed] [Google Scholar]
  5. Buchberger A., Schröder H., Büttner M., Valencia A., Bukau B. A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Nat Struct Biol. 1994 Feb;1(2):95–101. doi: 10.1038/nsb0294-95. [DOI] [PubMed] [Google Scholar]
  6. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eaton T., Shearman C., Gasson M. Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis. J Gen Microbiol. 1993 Dec;139(12):3253–3264. doi: 10.1099/00221287-139-12-3253. [DOI] [PubMed] [Google Scholar]
  8. Engel J. N., Pollack J., Perara E., Ganem D. Heat shock response of murine Chlamydia trachomatis. J Bacteriol. 1990 Dec;172(12):6959–6972. doi: 10.1128/jb.172.12.6959-6972.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feng D. F., Doolittle R. F. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. doi: 10.1007/BF02603120. [DOI] [PubMed] [Google Scholar]
  10. Gamer J., Bujard H., Bukau B. Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell. 1992 May 29;69(5):833–842. doi: 10.1016/0092-8674(92)90294-m. [DOI] [PubMed] [Google Scholar]
  11. Georgopoulos C. P. Bacterial mutants in which the gene N function of bacteriophage lambda is blocked have an altered RNA polymerase. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2977–2981. doi: 10.1073/pnas.68.12.2977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Georgopoulos C. The emergence of the chaperone machines. Trends Biochem Sci. 1992 Aug;17(8):295–299. doi: 10.1016/0968-0004(92)90439-g. [DOI] [PubMed] [Google Scholar]
  13. Georgopoulos C., Welch W. J. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol. 1993;9:601–634. doi: 10.1146/annurev.cb.09.110193.003125. [DOI] [PubMed] [Google Scholar]
  14. Higgins D. G., Sharp P. M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr;5(2):151–153. doi: 10.1093/bioinformatics/5.2.151. [DOI] [PubMed] [Google Scholar]
  15. Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ikeda E., Yoshida S., Mitsuzawa H., Uno I., Toh-e A. YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions. FEBS Lett. 1994 Feb 21;339(3):265–268. doi: 10.1016/0014-5793(94)80428-1. [DOI] [PubMed] [Google Scholar]
  17. Johnson C., Chandrasekhar G. N., Georgopoulos C. Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro. J Bacteriol. 1989 Mar;171(3):1590–1596. doi: 10.1128/jb.171.3.1590-1596.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kawula T. H., Lelivelt M. J. Mutations in a gene encoding a new Hsp70 suppress rapid DNA inversion and bgl activation, but not proU derepression, in hns-1 mutant Escherichia coli. J Bacteriol. 1994 Feb;176(3):610–619. doi: 10.1128/jb.176.3.610-619.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  21. Liberek K., Georgopoulos C. Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11019–11023. doi: 10.1073/pnas.90.23.11019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liberek K., Marszalek J., Ang D., Georgopoulos C., Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2874–2878. doi: 10.1073/pnas.88.7.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liberek K., Skowyra D., Zylicz M., Johnson C., Georgopoulos C. The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J Biol Chem. 1991 Aug 5;266(22):14491–14496. [PubMed] [Google Scholar]
  24. Lipinska B., King J., Ang D., Georgopoulos C. Sequence analysis and transcriptional regulation of the Escherichia coli grpE gene, encoding a heat shock protein. Nucleic Acids Res. 1988 Aug 11;16(15):7545–7562. doi: 10.1093/nar/16.15.7545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  26. Mensa-Wilmot K., Seaby R., Alfano C., Wold M. C., Gomes B., McMacken R. Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. J Biol Chem. 1989 Feb 15;264(5):2853–2861. [PubMed] [Google Scholar]
  27. Narberhaus F., Giebeler K., Bahl H. Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J Bacteriol. 1992 May;174(10):3290–3299. doi: 10.1128/jb.174.10.3290-3299.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Osipiuk J., Georgopoulos C., Zylicz M. Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein. J Biol Chem. 1993 Mar 5;268(7):4821–4827. [PubMed] [Google Scholar]
  29. Palleros D. R., Reid K. L., Shi L., Welch W. J., Fink A. L. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis. Nature. 1993 Oct 14;365(6447):664–666. doi: 10.1038/365664a0. [DOI] [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Phillips G. J., Silhavy T. J. Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli. Nature. 1990 Apr 26;344(6269):882–884. doi: 10.1038/344882a0. [DOI] [PubMed] [Google Scholar]
  32. Saito H., Nakamura Y., Uchida H. A transducing lambda phage carrying grpE, a bacterial gene necessary for lambda DNA replication, and two ribosomal protein genes, rpsP (S16) and rplS (L19). Mol Gen Genet. 1978 Oct 24;165(3):247–256. doi: 10.1007/BF00332523. [DOI] [PubMed] [Google Scholar]
  33. Saito H., Uchida H. Initiation of the DNA replication of bacteriophage lambda in Escherichia coli K12. J Mol Biol. 1977 Jun 15;113(1):1–25. doi: 10.1016/0022-2836(77)90038-9. [DOI] [PubMed] [Google Scholar]
  34. Schröder H., Langer T., Hartl F. U., Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 1993 Nov;12(11):4137–4144. doi: 10.1002/j.1460-2075.1993.tb06097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seaton B. L., Vickery L. E. A gene encoding a DnaK/hsp70 homolog in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2066–2070. doi: 10.1073/pnas.91.6.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sherman MYu, Goldberg A. L. Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli. EMBO J. 1992 Jan;11(1):71–77. doi: 10.1002/j.1460-2075.1992.tb05029.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Skowyra D., Georgopoulos C., Zylicz M. The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell. 1990 Sep 7;62(5):939–944. doi: 10.1016/0092-8674(90)90268-j. [DOI] [PubMed] [Google Scholar]
  38. Straus D., Walter W., Gross C. A. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 1990 Dec;4(12A):2202–2209. doi: 10.1101/gad.4.12a.2202. [DOI] [PubMed] [Google Scholar]
  39. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tilly K., Hauser R., Campbell J., Ostheimer G. J. Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants. Mol Microbiol. 1993 Feb;7(3):359–369. doi: 10.1111/j.1365-2958.1993.tb01128.x. [DOI] [PubMed] [Google Scholar]
  41. Tilly K., McKittrick N., Zylicz M., Georgopoulos C. The dnaK protein modulates the heat-shock response of Escherichia coli. Cell. 1983 Sep;34(2):641–646. doi: 10.1016/0092-8674(83)90396-3. [DOI] [PubMed] [Google Scholar]
  42. Ueguchi C., Kakeda M., Yamada H., Mizuno T. An analogue of the DnaJ molecular chaperone in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1054–1058. doi: 10.1073/pnas.91.3.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Voos W., Gambill B. D., Laloraya S., Ang D., Craig E. A., Pfanner N. Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes. Mol Cell Biol. 1994 Oct;14(10):6627–6634. doi: 10.1128/mcb.14.10.6627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wang R. F., Kushner S. R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed] [Google Scholar]
  45. Wetzstein M., Schumann W. Nucleotide sequence of a Bacillus subtilis gene homologous to the grpE gene of E. coli located immediately upstream of the dnaK gene. Nucleic Acids Res. 1990 Mar 11;18(5):1289–1289. doi: 10.1093/nar/18.5.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wild J., Kamath-Loeb A., Ziegelhoffer E., Lonetto M., Kawasaki Y., Gross C. A. Partial loss of function mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, affect highly conserved amino acids implicated in ATP binding and hydrolysis. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7139–7143. doi: 10.1073/pnas.89.15.7139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ziemienowicz A., Skowyra D., Zeilstra-Ryalls J., Fayet O., Georgopoulos C., Zylicz M. Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity. J Biol Chem. 1993 Dec 5;268(34):25425–25431. [PubMed] [Google Scholar]
  48. Zylicz M., Ang D., Georgopoulos C. The grpE protein of Escherichia coli. Purification and properties. J Biol Chem. 1987 Dec 25;262(36):17437–17442. [PubMed] [Google Scholar]
  49. Zylicz M., Ang D., Liberek K., Georgopoulos C. Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins. EMBO J. 1989 May;8(5):1601–1608. doi: 10.1002/j.1460-2075.1989.tb03544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zylicz M., Georgopoulos C. Purification and properties of the Escherichia coli dnaK replication protein. J Biol Chem. 1984 Jul 25;259(14):8820–8825. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES