Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Sep;177(17):4969–4973. doi: 10.1128/jb.177.17.4969-4973.1995

Multicopy suppression of cold-sensitive sec mutations in Escherichia coli.

P N Danese 1, C K Murphy 1, T J Silhavy 1
PMCID: PMC177272  PMID: 7665473

Abstract

Mutations in the secretory (sec) genes in Escherichia coli compromise protein translocation across the inner membrane and often confer conditional-lethal phenotypes. We have found that overproduction of the chaperonins GroES and GroEL from a multicopy plasmid suppresses a wide array of cold-sensitive sec mutations in E. coli. Suppression is accompanied by a stimulation of precursor protein translocation. This multicopy suppression does not bypass the Sec pathway because a deletion of secE is not suppressed under these conditions. Surprisingly, progressive deletion of the groE operon does not completely abolish the ability to suppress, indicating that the multicopy suppression of cold-sensitive sec mutations is not dependent on a functional groE operon. Indeed, overproduction of proteins unrelated to the process of protein export suppresses the secE501 cold-sensitive mutation, suggesting that protein overproduction, in and of itself, can confer mutations which compromise protein synthesis and the observation that low levels of protein synthesis inhibitors can suppress as well. In all cases, the mechanism of suppression is unrelated to the process of protein export. We suggest that the multicopy plasmids also suppress the sec mutations by compromising protein synthesis.

Full Text

The Full Text of this article is available as a PDF (273.9 KB).

Selected References

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

  1. Baba T., Jacq A., Brickman E., Beckwith J., Taura T., Ueguchi C., Akiyama Y., Ito K. Characterization of cold-sensitive secY mutants of Escherichia coli. J Bacteriol. 1990 Dec;172(12):7005–7010. doi: 10.1128/jb.172.12.7005-7010.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berman M. L., Beckwith J. Use of gene fusions to isolate promoter mutants in the transfer RNA gene tyrT of Escherichia coli. J Mol Biol. 1979 May 25;130(3):303–315. doi: 10.1016/0022-2836(79)90543-6. [DOI] [PubMed] [Google Scholar]
  3. Bieker K. L., Phillips G. J., Silhavy T. J. The sec and prl genes of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):291–310. doi: 10.1007/BF00763169. [DOI] [PubMed] [Google Scholar]
  4. Bieker K. L., Silhavy T. J. PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli. Cell. 1990 Jun 1;61(5):833–842. doi: 10.1016/0092-8674(90)90193-i. [DOI] [PubMed] [Google Scholar]
  5. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  6. Brickman E. R., Oliver D. B., Garwin J. L., Kumamoto C., Beckwith J. The use of extragenic suppressors to define genes involved in protein export in Escherichia coli. Mol Gen Genet. 1984;196(1):24–27. doi: 10.1007/BF00334087. [DOI] [PubMed] [Google Scholar]
  7. Dennis P. P. Effects of chloramphenicol on the transcriptional activities of ribosomal RNA and ribosomal protein genes in Escherichia coli. J Mol Biol. 1976 Dec 15;108(3):535–546. doi: 10.1016/s0022-2836(76)80135-0. [DOI] [PubMed] [Google Scholar]
  8. Dong H., Nilsson L., Kurland C. G. Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J Bacteriol. 1995 Mar;177(6):1497–1504. doi: 10.1128/jb.177.6.1497-1504.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Esnault Y., Blondel M. O., Deshaies R. J., Scheckman R., Képès F. The yeast SSS1 gene is essential for secretory protein translocation and encodes a conserved protein of the endoplasmic reticulum. EMBO J. 1993 Nov;12(11):4083–4093. doi: 10.1002/j.1460-2075.1993.tb06092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Ferro-Novick S., Honma M., Beckwith J. The product of gene secC is involved in the synthesis of exported proteins in E. coli. Cell. 1984 Aug;38(1):211–217. doi: 10.1016/0092-8674(84)90542-7. [DOI] [PubMed] [Google Scholar]
  12. Gardel C., Benson S., Hunt J., Michaelis S., Beckwith J. secD, a new gene involved in protein export in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1286–1290. doi: 10.1128/jb.169.3.1286-1290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gardel C., Johnson K., Jacq A., Beckwith J. The secD locus of E.coli codes for two membrane proteins required for protein export. EMBO J. 1990 Oct;9(10):3209–3216. doi: 10.1002/j.1460-2075.1990.tb07519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Huisman G. W., Kolter R. Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli. Science. 1994 Jul 22;265(5171):537–539. doi: 10.1126/science.7545940. [DOI] [PubMed] [Google Scholar]
  16. Kiino D. R., Silhavy T. J. Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli. J Bacteriol. 1984 Jun;158(3):878–883. doi: 10.1128/jb.158.3.878-883.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Knauf U., Jakob U., Engel K., Buchner J., Gaestel M. Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance. EMBO J. 1994 Jan 1;13(1):54–60. doi: 10.1002/j.1460-2075.1994.tb06234.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuldell N., Hochschild A. Amino acid substitutions in the -35 recognition motif of sigma 70 that result in defects in phage lambda repressor-stimulated transcription. J Bacteriol. 1994 May;176(10):2991–2998. doi: 10.1128/jb.176.10.2991-2998.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee C. A., Beckwith J. Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol. 1986 Jun;166(3):878–883. doi: 10.1128/jb.166.3.878-883.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Murphy C. K., Beckwith J. Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2557–2561. doi: 10.1073/pnas.91.7.2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy C. K., Stewart E. J., Beckwith J. A double counter-selection system for the study of null alleles of essential genes in Escherichia coli. Gene. 1995 Mar 21;155(1):1–7. doi: 10.1016/0378-1119(94)00920-n. [DOI] [PubMed] [Google Scholar]
  22. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  23. Oliver D. B. Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli. J Bacteriol. 1985 Jan;161(1):285–291. doi: 10.1128/jb.161.1.285-291.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pogliano K. J., Beckwith J. The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself. Genetics. 1993 Apr;133(4):763–773. doi: 10.1093/genetics/133.4.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Riggs P. D., Derman A. I., Beckwith J. A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics. 1988 Apr;118(4):571–579. doi: 10.1093/genetics/118.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sakaguchi M., Ueguchi C., Ito K., Omura T. Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli. J Biochem. 1991 Jun;109(6):799–802. doi: 10.1093/oxfordjournals.jbchem.a123460. [DOI] [PubMed] [Google Scholar]
  27. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  28. Schatz P. J., Bieker K. L., Ottemann K. M., Silhavy T. J., Beckwith J. One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery. EMBO J. 1991 Jul;10(7):1749–1757. doi: 10.1002/j.1460-2075.1991.tb07699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schatz P. J., Riggs P. D., Jacq A., Fath M. J., Beckwith J. The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev. 1989 Jul;3(7):1035–1044. doi: 10.1101/gad.3.7.1035. [DOI] [PubMed] [Google Scholar]
  30. Schmidt M. G., Rollo E. E., Grodberg J., Oliver D. B. Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli. J Bacteriol. 1988 Aug;170(8):3404–3414. doi: 10.1128/jb.170.8.3404-3414.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shiba K., Ito K., Nakamura Y., Dondon J., Grunberg-Manago M. Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli. EMBO J. 1986 Nov;5(11):3001–3006. doi: 10.1002/j.1460-2075.1986.tb04598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shiba K., Ito K., Yura T., Cerretti D. P. A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J. 1984 Mar;3(3):631–635. doi: 10.1002/j.1460-2075.1984.tb01859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Taura T., Akiyama Y., Ito K. Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes. Mol Gen Genet. 1994 May 10;243(3):261–269. doi: 10.1007/BF00301061. [DOI] [PubMed] [Google Scholar]
  35. Ueguchi C., Ito K. Multicopy suppression: an approach to understanding intracellular functioning of the protein export system. J Bacteriol. 1992 Mar;174(5):1454–1461. doi: 10.1128/jb.174.5.1454-1461.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Zeilstra-Ryalls J., Fayet O., Georgopoulos C. The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol. 1991;45:301–325. doi: 10.1146/annurev.mi.45.100191.001505. [DOI] [PubMed] [Google Scholar]

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

RESOURCES