Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 Dec;9(12):4027–4036. doi: 10.1002/j.1460-2075.1990.tb07624.x

Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone.

B Bukau 1, G C Walker 1
PMCID: PMC552175  PMID: 2249663

Abstract

An Escherichia coli mutant lacking HSP70 function, delta dnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30 degrees C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells. In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of delta dnaK52 mutants at 30 degrees C and also permit growth at low temperature. Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (sigma 32) of RNA polymerase. The sidB mutations affected four different regions of the sigma 32 protein and, in one case, resulted in a several fold reduction in the cellular concentration of sigma 32. Presence of any of the sidB mutations in delta dnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30 degrees C and decreased induction of the heat shock response after shift to 43.5 degrees C. These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.

Full text

PDF
4031

Images in this article

4028Fig. 1.
on p.4028
4029Fig. 2.
on p.4029
4030Fig. 4.
on p.4030
4031Fig. 5.
on p.4031
4031Fig. 6.
on p.4031
4032Fig. 7.
on p.4032
4032Fig. 8.
on p.4032

Selected References

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

  1. Bahl H., Echols H., Straus D. B., Court D., Crowl R., Georgopoulos C. P. Induction of the heat shock response of E. coli through stabilization of sigma 32 by the phage lambda cIII protein. Genes Dev. 1987 Mar;1(1):57–64. doi: 10.1101/gad.1.1.57. [DOI] [PubMed] [Google Scholar]
  2. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bloom M., Skelly S., VanBogelen R., Neidhardt F., Brot N., Weissbach H. In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes. J Bacteriol. 1986 May;166(2):380–384. doi: 10.1128/jb.166.2.380-384.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bukau B., Walker G. C. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J Bacteriol. 1989 May;171(5):2337–2346. doi: 10.1128/jb.171.5.2337-2346.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bukau B., Walker G. C. Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures. J Bacteriol. 1989 Nov;171(11):6030–6038. doi: 10.1128/jb.171.11.6030-6038.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chiang H. L., Terlecky S. R., Plant C. P., Dice J. F. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science. 1989 Oct 20;246(4928):382–385. doi: 10.1126/science.2799391. [DOI] [PubMed] [Google Scholar]
  7. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  8. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Craig E. A., Kramer J., Kosic-Smithers J. SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4156–4160. doi: 10.1073/pnas.84.12.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  11. Doi R. H., Wang L. F. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol Rev. 1986 Sep;50(3):227–243. doi: 10.1128/mr.50.3.227-243.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Echols H. Multiple DNA-protein interactions governing high-precision DNA transactions. Science. 1986 Sep 5;233(4768):1050–1056. doi: 10.1126/science.2943018. [DOI] [PubMed] [Google Scholar]
  13. Ellis J. Proteins as molecular chaperones. 1987 Jul 30-Aug 5Nature. 328(6129):378–379. doi: 10.1038/328378a0. [DOI] [PubMed] [Google Scholar]
  14. Funnell B. E. Mini-P1 plasmid partitioning: excess ParB protein destabilizes plasmids containing the centromere parS. J Bacteriol. 1988 Feb;170(2):954–960. doi: 10.1128/jb.170.2.954-960.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goff S. A., Goldberg A. L. An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli. J Biol Chem. 1987 Apr 5;262(10):4508–4515. [PubMed] [Google Scholar]
  16. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  17. Grossman A. D., Straus D. B., Walter W. A., Gross C. A. Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev. 1987 Apr;1(2):179–184. doi: 10.1101/gad.1.2.179. [DOI] [PubMed] [Google Scholar]
  18. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  19. Herendeen S. L., VanBogelen R. A., Neidhardt F. C. Levels of major proteins of Escherichia coli during growth at different temperatures. J Bacteriol. 1979 Jul;139(1):185–194. doi: 10.1128/jb.139.1.185-194.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Itikawa H., Ryu J. Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. J Bacteriol. 1979 May;138(2):339–344. doi: 10.1128/jb.138.2.339-344.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kusukawa N., Yura T. Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes Dev. 1988 Jul;2(7):874–882. doi: 10.1101/gad.2.7.874. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Landick R., Vaughn V., Lau E. T., VanBogelen R. A., Erickson J. W., Neidhardt F. C. Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor. Cell. 1984 Aug;38(1):175–182. doi: 10.1016/0092-8674(84)90538-5. [DOI] [PubMed] [Google Scholar]
  25. Lemaux P. G., Herendeen S. L., Bloch P. L., Neidhardt F. C. Transient rates of synthesis of individual polypeptides in E. coli following temperature shifts. Cell. 1978 Mar;13(3):427–434. doi: 10.1016/0092-8674(78)90317-3. [DOI] [PubMed] [Google Scholar]
  26. Liberek K., Georgopoulos C., Zylicz M. Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6632–6636. doi: 10.1073/pnas.85.18.6632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  28. Paek K. H., Walker G. C. Defect in expression of heat-shock proteins at high temperature in xthA mutants. J Bacteriol. 1986 Mar;165(3):763–770. doi: 10.1128/jb.165.3.763-770.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paek K. H., Walker G. C. Escherichia coli dnaK null mutants are inviable at high temperature. J Bacteriol. 1987 Jan;169(1):283–290. doi: 10.1128/jb.169.1.283-290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parsell D. A., Silber K. R., Sauer R. T. Carboxy-terminal determinants of intracellular protein degradation. Genes Dev. 1990 Feb;4(2):277–286. doi: 10.1101/gad.4.2.277. [DOI] [PubMed] [Google Scholar]
  31. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  32. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  33. Rothman J. E., Schmid S. L. Enzymatic recycling of clathrin from coated vesicles. Cell. 1986 Jul 4;46(1):5–9. doi: 10.1016/0092-8674(86)90852-4. [DOI] [PubMed] [Google Scholar]
  34. Sakakibara Y. The dnaK gene of Escherichia coli functions in initiation of chromosome replication. J Bacteriol. 1988 Feb;170(2):972–979. doi: 10.1128/jb.170.2.972-979.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sell S. M., Eisen C., Ang D., Zylicz M., Georgopoulos C. Isolation and characterization of dnaJ null mutants of Escherichia coli. J Bacteriol. 1990 Sep;172(9):4827–4835. doi: 10.1128/jb.172.9.4827-4835.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Siegele D. A., Hu J. C., Walter W. A., Gross C. A. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol. 1989 Apr 20;206(4):591–603. doi: 10.1016/0022-2836(89)90568-8. [DOI] [PubMed] [Google Scholar]
  38. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sternberg N., Austin S. Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L. J Bacteriol. 1983 Feb;153(2):800–812. doi: 10.1128/jb.153.2.800-812.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Straus D. B., Walter W. A., Gross C. A. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 1988 Dec;2(12B):1851–1858. doi: 10.1101/gad.2.12b.1851. [DOI] [PubMed] [Google Scholar]
  41. Straus D. B., Walter W. A., Gross C. A. The heat shock response of E. coli is regulated by changes in the concentration of sigma 32. Nature. 1987 Sep 24;329(6137):348–351. doi: 10.1038/329348a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Tilly K., Spence J., Georgopoulos C. Modulation of stability of the Escherichia coli heat shock regulatory factor sigma. J Bacteriol. 1989 Mar;171(3):1585–1589. doi: 10.1128/jb.171.3.1585-1589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tilly K., Yarmolinsky M. Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication. J Bacteriol. 1989 Nov;171(11):6025–6029. doi: 10.1128/jb.171.11.6025-6029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wickner S. H. Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2690–2694. doi: 10.1073/pnas.87.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhou Y. N., Kusukawa N., Erickson J. W., Gross C. A., Yura T. Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32. J Bacteriol. 1988 Aug;170(8):3640–3649. doi: 10.1128/jb.170.8.3640-3649.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Zylicz M., LeBowitz J. H., McMacken R., Georgopoulos C. The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6431–6435. doi: 10.1073/pnas.80.21.6431. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES