Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Nov;176(22):6980–6985. doi: 10.1128/jb.176.22.6980-6985.1994

A carboxy-terminal deletion impairs the assembly of GroEL and confers a pleiotropic phenotype in Escherichia coli K-12.

B P Burnett 1, A L Horwich 1, K B Low 1
PMCID: PMC197070  PMID: 7961461

Abstract

A series of COOH-terminal deletions of the chaperonin GroEL have been examined for effects in vivo at haploid copy number on the essential requirement of GroEL for cell growth. Strains with a deletion of up to 27 COOH-terminal amino acids were viable, but not viable strain could be isolated with a deletion of 28 or more codons. When substitutions were placed in the COOH-terminal amino acid Val-521 of the 27-amino-acid-deleted (delta 27) mutant, we found variable effect--Trp and Glu led to inviability, whereas Arg and Gly were viable but slow growing. The effects of the Arg substitution plus deletion (V521R delta) were examined in more detail. Whereas the delta 27 mutant with the wild-type residue Val-521 grew as well as a strain with wild-type GroEL, the V521R delta mutant strain (groEL202) exhibited a broad range of phenotypic defects. These include slow growth; filamentous morphology; a defect in plating lambda; absence of activity of expressed human ornithine transcarbamylase, as seen in other GroEL mutants; and several newly observed defects, such as absence of motility, sensitivity to UV light and mitomycin, a defect in one mode of specialized transduction, and inability to grow on rhamnose. Sucrose gradient analysis of extracts from the V521R delta cells showed a substantially reduced level of GroEL sedimenting at the normal 20S position of the assembled tetradecamer and a relatively large amount of more lightly sedimenting subunits. This indicates that the substitution-deletion mutation interferes with oligomeric assembly of GroEL into its functional form. This is discussed in light of the recently determined crystal structure of GroEL.

Full text

PDF
6982

Images in this article

6984Image
on p.6984

Selected References

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

  1. Bagg A., Kenyon C. J., Walker G. C. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5749–5753. doi: 10.1073/pnas.78.9.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergmans H. E., Hoekstra W. P., Zuidweg E. M. Conjugation in Escherichia coli: a study of recombination and the fate of donor DNA at the level of the zygote. Mol Gen Genet. 1975;137(1):1–10. doi: 10.1007/BF00332536. [DOI] [PubMed] [Google Scholar]
  3. Birge E. A., Low K. B. Detection of transcribable recombination products following conjugation in rec+, reCB- and recC-strains of Escherichia coli K12. J Mol Biol. 1974 Mar 15;83(4):447–457. doi: 10.1016/0022-2836(74)90506-3. [DOI] [PubMed] [Google Scholar]
  4. Coppo A., Manzi A., Pulitzer J. F., Takahashi H. Abortive bacteriophage T4 head assembly in mutants of Escherichia coli. J Mol Biol. 1973 May 5;76(1):61–87. doi: 10.1016/0022-2836(73)90081-8. [DOI] [PubMed] [Google Scholar]
  5. Donnelly C. E., Walker G. C. Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants. J Bacteriol. 1992 May;174(10):3133–3139. doi: 10.1128/jb.174.10.3133-3139.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donnelly C. E., Walker G. C. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis. J Bacteriol. 1989 Nov;171(11):6117–6125. doi: 10.1128/jb.171.11.6117-6125.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fayet O., Louarn J. M., Georgopoulos C. Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol Gen Genet. 1986 Mar;202(3):435–445. doi: 10.1007/BF00333274. [DOI] [PubMed] [Google Scholar]
  8. Fayet O., Ziegelhoffer T., Georgopoulos C. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J Bacteriol. 1989 Mar;171(3):1379–1385. doi: 10.1128/jb.171.3.1379-1385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Georgopoulos C. P., Eisen H. Bacterial mutants which block phage assembly. J Supramol Struct. 1974;2(2-4):349–359. doi: 10.1002/jss.400020224. [DOI] [PubMed] [Google Scholar]
  10. Georgopoulos C. P., Hendrix R. W., Kaiser A. D., Wood W. B. Role of the host cell in bacteriophage morphogenesis: effects of a bacterial mutation on T4 head assembly. Nat New Biol. 1972 Sep 13;239(89):38–41. doi: 10.1038/newbio239038a0. [DOI] [PubMed] [Google Scholar]
  11. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  12. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  13. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  14. Horwich A. L., Low K. B., Fenton W. A., Hirshfield I. N., Furtak K. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell. 1993 Sep 10;74(5):909–917. doi: 10.1016/0092-8674(93)90470-b. [DOI] [PubMed] [Google Scholar]
  15. Horwich A. L., Willison K. R. Protein folding in the cell: functions of two families of molecular chaperone, hsp 60 and TF55-TCP1. Philos Trans R Soc Lond B Biol Sci. 1993 Mar 29;339(1289):313–326. doi: 10.1098/rstb.1993.0030. [DOI] [PubMed] [Google Scholar]
  16. Howard-Flanders P., Theriot L., Stedeford J. B. Some properties of excision-defective recombination-deficient mutants of Escherichia coli K-12. J Bacteriol. 1969 Mar;97(3):1134–1141. doi: 10.1128/jb.97.3.1134-1141.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jenkins A. J., March J. B., Oliver I. R., Masters M. A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene. Mol Gen Genet. 1986 Mar;202(3):446–454. doi: 10.1007/BF00333275. [DOI] [PubMed] [Google Scholar]
  18. Kalousek F., François B., Rosenberg L. E. Isolation and characterization of ornithine transcarbamylase from normal human liver. J Biol Chem. 1978 Jun 10;253(11):3939–3944. [PubMed] [Google Scholar]
  19. Kanemori M., Mori H., Yura T. Effects of reduced levels of GroE chaperones on protein metabolism: enhanced synthesis of heat shock proteins during steady-state growth of Escherichia coli. J Bacteriol. 1994 Jul;176(14):4235–4242. doi: 10.1128/jb.176.14.4235-4242.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kato T., Shinoura Y. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol Gen Genet. 1977 Nov 14;156(2):121–131. doi: 10.1007/BF00283484. [DOI] [PubMed] [Google Scholar]
  21. Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  23. Krueger J. H., Walker G. C. groEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1499–1503. doi: 10.1073/pnas.81.5.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Kusukawa N., Yura T., Ueguchi C., Akiyama Y., Ito K. Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J. 1989 Nov;8(11):3517–3521. doi: 10.1002/j.1460-2075.1989.tb08517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laine P. S., Meyer R. R. Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46. J Bacteriol. 1992 May;174(10):3204–3211. doi: 10.1128/jb.174.10.3204-3211.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liu S. K., Tessman I. groE genes affect SOS repair in Escherichia coli. J Bacteriol. 1990 Oct;172(10):6135–6138. doi: 10.1128/jb.172.10.6135-6138.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Low B. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J Bacteriol. 1973 Feb;113(2):798–812. doi: 10.1128/jb.113.2.798-812.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McLennan N. F., Girshovich A. S., Lissin N. M., Charters Y., Masters M. The strongly conserved carboxyl-terminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable. Mol Microbiol. 1993 Jan;7(1):49–58. doi: 10.1111/j.1365-2958.1993.tb01096.x. [DOI] [PubMed] [Google Scholar]
  30. Miki T., Orita T., Furuno M., Horiuchi T. Control of cell division by sex factor F in Escherichia coli. III. Participation of the groES (mopB) gene of the host bacteria. J Mol Biol. 1988 May 20;201(2):327–338. doi: 10.1016/0022-2836(88)90141-6. [DOI] [PubMed] [Google Scholar]
  31. Porter R. D., Lark M. W., Low K. B. Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda. J Virol. 1981 May;38(2):497–503. doi: 10.1128/jvi.38.2.497-503.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Porter R. D., McLaughlin T., Low B. Transduction versus "conjuduction": evidence for multiple roles for exonuclease V in genetic recombination in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1043–1047. doi: 10.1101/sqb.1979.043.01.113. [DOI] [PubMed] [Google Scholar]
  33. Porter R. D. Specialized transduction with lambda plac5: involvement of the RecE and RecF recombination pathways. Genetics. 1983 Oct;105(2):247–257. doi: 10.1093/genetics/105.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ruben S. M., VanDenBrink-Webb S. E., Rein D. C., Meyer R. R. Suppression of the Escherichia coli ssb-1 mutation by an allele of groEL. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3767–3771. doi: 10.1073/pnas.85.11.3767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schellhorn H. E., Low K. B. Indirect stimulation of recombination in Escherichia coli K-12: dependence on recJ, uvrA, and uvrD. J Bacteriol. 1991 Oct;173(19):6192–6198. doi: 10.1128/jb.173.19.6192-6198.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Steinborn G. Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. I. Isolation of uvm mutants and their phenotypical characterization in DNA repair and mutagenesis. Mol Gen Genet. 1978 Sep 20;165(1):87–93. doi: 10.1007/BF00270380. [DOI] [PubMed] [Google Scholar]
  37. Sternberg N. Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization. J Mol Biol. 1973 May 5;76(1):1–23. doi: 10.1016/0022-2836(73)90078-8. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Takano T., Kakefuda T. Involvement of a bacterial factor in morphogenesis of bacteriophage capsid. Nat New Biol. 1972 Sep 13;239(89):34–37. doi: 10.1038/newbio239034a0. [DOI] [PubMed] [Google Scholar]
  40. Tilly K., Murialdo H., Georgopoulos C. Identification of a second Escherichia coli groE gene whose product is necessary for bacteriophage morphogenesis. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1629–1633. doi: 10.1073/pnas.78.3.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Dyk T. K., Gatenby A. A., LaRossa R. A. Demonstration by genetic suppression of interaction of GroE products with many proteins. Nature. 1989 Nov 23;342(6248):451–453. doi: 10.1038/342451a0. [DOI] [PubMed] [Google Scholar]
  42. Van Houten B. Nucleotide excision repair in Escherichia coli. Microbiol Rev. 1990 Mar;54(1):18–51. doi: 10.1128/mr.54.1.18-51.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wada M., Itikawa H. Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA. J Bacteriol. 1984 Feb;157(2):694–696. doi: 10.1128/jb.157.2.694-696.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zweig M., Cummings D. J. Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein. J Mol Biol. 1973 Nov 5;80(3):505–518. doi: 10.1016/0022-2836(73)90418-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES