Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Nov;169(11):5201–5208. doi: 10.1128/jb.169.11.5201-5208.1987

Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit.

L M Guzman-Verduzco 1, Y M Kupersztoch 1
PMCID: PMC213927  PMID: 3312169

Abstract

The 3' terminus of the DNA coding for the extracellular Escherichia coli heat-stable enterotoxin (ST) devoid of transcription and translation stop signals was fused to the 5' terminus of the DNA coding for the periplasmic B subunit of the heat-labile enterotoxin (LTB) deleted of ribosomal binding sites and leader peptide. By RNA-DNA hybridization analysis, it was shown that the fused DNA was transcribed in vivo into an RNA species in close agreement with the expected molecular weight inferred from the nucleotide sequence. The translation products of the fused DNA resulted in a hybrid molecule recognized in Western blots (immunoblots) with antibodies directed against the heat-labile moiety. Anti-LTB antibodies coupled to a solid support bound ST and LTB simultaneously when incubated with ST-LTB cellular extracts. By [35S]cysteine pulse-chase experiments, it was shown that the fused ST-LTB polypeptide was converted from a precursor with an equivalent electrophoretic mobility of 20,800 daltons to an approximately 18,500-dalton species, which accumulated within the cell. The data suggest that wild-type ST undergoes at least two processing steps during its export to the culture supernatant. Blocking the natural carboxy terminus of ST inhibited the second proteolytic step and extracellular delivery of the hybrid molecule.

Full text

PDF
5201

Images in this article

5204Image
on p.5204
5204Image
on p.5204
5205Image
on p.5205
5206Image
on p.5206

Selected References

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

  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Aimoto S., Takao T., Shimonishi Y., Hara S., Takeda T., Takeda Y., Miwatani T. Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli. Eur J Biochem. 1982 Dec 15;129(2):257–263. doi: 10.1111/j.1432-1033.1982.tb07047.x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clements J. D., Finkelstein R. A. Immunological cross-reactivity between a heat-labile enterotoxin(s) of Escherichia coli and subunits of Vibrio cholerae enterotoxin. Infect Immun. 1978 Sep;21(3):1036–1039. doi: 10.1128/iai.21.3.1036-1039.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clements J. D., Finkelstein R. A. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. Infect Immun. 1979 Jun;24(3):760–769. doi: 10.1128/iai.24.3.760-769.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dallas W. S., Gill D. M., Falkow S. Cistrons encoding Escherichia coli heat-labile toxin. J Bacteriol. 1979 Sep;139(3):850–858. doi: 10.1128/jb.139.3.850-858.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dean A. G., Ching Y. C., Williams R. G., Harden L. B. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J Infect Dis. 1972 Apr;125(4):407–411. doi: 10.1093/infdis/125.4.407. [DOI] [PubMed] [Google Scholar]
  10. Geary S. J., Marchlewicz B. A., Finkelstein R. A. Comparison of heat-labile enterotoxins from porcine and human strains of Escherichia coli. Infect Immun. 1982 Apr;36(1):215–220. doi: 10.1128/iai.36.1.215-220.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gill D. M., Clements J. D., Robertson D. C., Finkelstein R. A. Subunit number and arrangement in Escherichia coli heat-labile enterotoxin. Infect Immun. 1981 Sep;33(3):677–682. doi: 10.1128/iai.33.3.677-682.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guerrant R. L., Kirchhoff L. V., Shields D. S., Nations M. K., Leslie J., de Sousa M. A., Araujo J. G., Correia L. L., Sauer K. T., McClelland K. E. Prospective study of diarrheal illnesses in northeastern Brazil: patterns of disease, nutritional impact, etiologies, and risk factors. J Infect Dis. 1983 Dec;148(6):986–997. doi: 10.1093/infdis/148.6.986. [DOI] [PubMed] [Google Scholar]
  13. Guzmán-Verduzco L. M., Fonseca R., Kupersztoch-Portnoy Y. M. Thermoactivation of a periplasmic heat-stable enterotoxin of Escherichia coli. J Bacteriol. 1983 Apr;154(1):146–151. doi: 10.1128/jb.154.1.146-151.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hofstra H., Witholt B. Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli. J Biol Chem. 1984 Dec 25;259(24):15182–15187. [PubMed] [Google Scholar]
  15. King T. C., Sirdeskmukh R., Schlessinger D. Nucleolytic processing of ribonucleic acid transcripts in procaryotes. Microbiol Rev. 1986 Dec;50(4):428–451. doi: 10.1128/mr.50.4.428-451.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klipstein F. A., Engert R. F., Houghten R. A. Immunisation of volunteers with a synthetic peptide vaccine for enterotoxigenic Escherichia coli. Lancet. 1986 Mar 1;1(8479):471–472. doi: 10.1016/s0140-6736(86)92930-2. [DOI] [PubMed] [Google Scholar]
  17. Kupersztoch Y. M., Helinski D. R. A catenated DNA molecule as an intermediate in the replication of the resistance transfer factor R6K in Escherichia coli. Biochem Biophys Res Commun. 1973 Oct 15;54(4):1451–1459. doi: 10.1016/0006-291x(73)91149-2. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. LURIA S. E., BURROUS J. W. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. doi: 10.1128/jb.74.4.461-476.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Martínez-Cadena M. G., Guzman-Verduzco L. M., Stieglitz H., Kupersztoch-Portnoy Y. M. Catabolite repression of Escherichia coli heat-stable enterotoxin activity. J Bacteriol. 1981 Feb;145(2):722–728. doi: 10.1128/jb.145.2.722-728.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  23. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  24. Moseley S. L., Hardy J. W., Hug M. I., Echeverria P., Falkow S. Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli. Infect Immun. 1983 Mar;39(3):1167–1174. doi: 10.1128/iai.39.3.1167-1174.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moss J., Osborne J. C., Jr, Fishman P. H., Nakaya S., Robertson D. C. Escherichia coli heat-labile enterotoxin. Ganglioside specificity and ADP-ribosyltransferase activity. J Biol Chem. 1981 Dec 25;256(24):12861–12865. [PubMed] [Google Scholar]
  26. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
  27. Palva E. T., Hirst T. R., Hardy S. J., Holmgren J., Randall L. Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli. J Bacteriol. 1981 Apr;146(1):325–330. doi: 10.1128/jb.146.1.325-330.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  29. Pickett C. L., Twiddy E. M., Belisle B. W., Holmes R. K. Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli. J Bacteriol. 1986 Feb;165(2):348–352. doi: 10.1128/jb.165.2.348-352.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith H. W., Gyles C. L. The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Escherichia coli of porcine origin. J Med Microbiol. 1970 Aug;3(3):387–401. doi: 10.1099/00222615-3-3-387. [DOI] [PubMed] [Google Scholar]
  33. So M., McCarthy B. J. Nucleotide sequence of the bacterial transposon Tn1681 encoding a heat-stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4011–4015. doi: 10.1073/pnas.77.7.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spicer E. K., Noble J. A. Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene. J Biol Chem. 1982 May 25;257(10):5716–5721. [PubMed] [Google Scholar]
  35. 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]
  36. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  37. Yamamoto T., Yokota T. Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans. J Bacteriol. 1983 Aug;155(2):728–733. doi: 10.1128/jb.155.2.728-733.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES