Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jun;179(11):3383–3390. doi: 10.1128/jb.179.11.3383-3390.1997

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

H Yamanaka 1, T Nomura 1, Y Fujii 1, K Okamoto 1
PMCID: PMC179126  PMID: 9171378

Abstract

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.

Full Text

The Full Text of this article is available as a PDF (979.5 KB).

Selected References

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

  1. 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]
  2. Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
  3. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. He S. Y., Schoedel C., Chatterjee A. K., Collmer A. Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane. J Bacteriol. 1991 Jul;173(14):4310–4317. doi: 10.1128/jb.173.14.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Holland I. B., Kenny B., Blight M. Haemolysin secretion from E coli. Biochimie. 1990 Feb-Mar;72(2-3):131–141. doi: 10.1016/0300-9084(90)90138-7. [DOI] [PubMed] [Google Scholar]
  6. Howard S. P., Buckley J. T. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol. 1985 Mar;161(3):1118–1124. doi: 10.1128/jb.161.3.1118-1124.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Lindeberg M., Collmer A. Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J Bacteriol. 1992 Nov;174(22):7385–7397. doi: 10.1128/jb.174.22.7385-7397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Létoffé S., Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990 May;9(5):1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Missiakas D., Georgopoulos C., Raina S. Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7084–7088. doi: 10.1073/pnas.90.15.7084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Okamoto K., Baba T., Yamanaka H., Akashi N., Fujii Y. Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II. J Bacteriol. 1995 Aug;177(16):4579–4586. doi: 10.1128/jb.177.16.4579-4586.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Okamoto K., Okamoto K., Yukitake J., Kawamoto Y., Miyama A. Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis. Infect Immun. 1987 Sep;55(9):2121–2125. doi: 10.1128/iai.55.9.2121-2125.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Okamoto K., Okamoto K., Yukitake J., Miyama A. Reduction of enterotoxic activity of Escherichia coli heat-stable enterotoxin by substitution for an asparagine residue. Infect Immun. 1988 Aug;56(8):2144–2148. doi: 10.1128/iai.56.8.2144-2148.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Okamoto K., Takahara M. Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion. J Bacteriol. 1990 Sep;172(9):5260–5265. doi: 10.1128/jb.172.9.5260-5265.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pugsley A. P., Reyss I. Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. Mol Microbiol. 1990 Mar;4(3):365–379. doi: 10.1111/j.1365-2958.1990.tb00604.x. [DOI] [PubMed] [Google Scholar]
  16. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rasheed J. K., Guzmán-Verduzco L. M., Kupersztoch Y. M. Two precursors of the heat-stable enterotoxin of Escherichia coli: evidence of extracellular processing. Mol Microbiol. 1990 Feb;4(2):265–273. doi: 10.1111/j.1365-2958.1990.tb00593.x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Staples S. J., Asher S. E., Giannella R. A. Purification and characterization of heat-stable enterotoxin produced by a strain of E. coli pathogenic for man. J Biol Chem. 1980 May 25;255(10):4716–4721. [PubMed] [Google Scholar]
  20. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  21. Takao T., Hitouji T., Aimoto S., Shimonishi Y., Hara S., Takeda T., Takeda Y., Miwatani T. Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D. FEBS Lett. 1983 Feb 7;152(1):1–5. doi: 10.1016/0014-5793(83)80469-4. [DOI] [PubMed] [Google Scholar]
  22. Wandersman C., Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. doi: 10.1073/pnas.87.12.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wong K. R., Green M. J., Buckley J. T. Extracellular secretion of cloned aerolysin and phospholipase by Aeromonas salmonicida. J Bacteriol. 1989 May;171(5):2523–2527. doi: 10.1128/jb.171.5.2523-2527.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yamanaka H., Fuke Y., Hitotsubashi S., Fujii Y., Okamoto K. Functional properties of pro region of Escherichia coli heat-stable enterotoxin. Microbiol Immunol. 1993;37(3):195–205. doi: 10.1111/j.1348-0421.1993.tb03200.x. [DOI] [PubMed] [Google Scholar]
  25. Yamanaka H., Kameyama M., Baba T., Fujii Y., Okamoto K. Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation. J Bacteriol. 1994 May;176(10):2906–2913. doi: 10.1128/jb.176.10.2906-2913.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yamanaka H., Okamoto K. Amino acid residues in the pro region of Escherichia coli heat-stable enterotoxin I that affect efficiency of translocation across the inner membrane. Infect Immun. 1996 Jul;64(7):2700–2708. doi: 10.1128/iai.64.7.2700-2708.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yang Y., Gao Z., Guzmán-Verduzco L. M., Tachias K., Kupersztoch Y. M. Secretion of the STA3 heat-stable enterotoxin of Escherichia coli: extracellular delivery of Pro-STA is accomplished by either Pro or STA. Mol Microbiol. 1992 Dec;6(23):3521–3529. doi: 10.1111/j.1365-2958.1992.tb01787.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES