Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 May;63(5):1767–1776. doi: 10.1128/iai.63.5.1767-1776.1995

A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli.

O G Gómez-Duarte 1, J B Kaper 1
PMCID: PMC173222  PMID: 7729884

Abstract

Enteropathogenic Escherichia coli (EPEC) organisms produce a characteristic histopathology in intestinal epithelial cells called attaching and effacing lesions. The eaeA gene is associated with attaching and effacing lesions and encodes intimin, a 94-kDa outer membrane protein. A 60-MDa plasmid, pMAR2, is essential for full virulence of EPEC strain E2348/69 (O127:H6). We have cloned sequences from pMAR2 that increase expression of the chromosomal eaeA gene as shown by increased alkaline phosphatase activity of an eaeA::TnphoA gene fusion, increased expression of the intimin protein, and increased production of eaeA mRNA. These sequences are called per for plasmid-encoded regulator. pMAR2-cured JPN15 containing cloned per sequences adheres to HEp-2 cells in greater numbers than JPN15 carrying the plasmid vector only. The cloned per sequences contain four open reading frames (ORFs) which have been designated perA through perD. Only perC can by itself activate expression of eaeA::TnphoA, although the levels of alkaline phosphatase activity seen with this ORF alone are considerably lower than those seen when all four ORFs are present. The molecular sizes of polypeptides predicted from perA, perB, perC, and perD ORFs are 24, 14.8, 10.5, and 9.4 kDa, respectively. The PerA predicted protein shares homology with members of the AraC family of bacterial regulators, but PerB, PerC, and PerD have no striking homology with previously described prokaryotic proteins. Our studies indicate that plasmid-encoded factors regulate the expression of eaeA and possibly genes encoding other outer membrane proteins and may be important for virulence of EPEC.

Full Text

The Full Text of this article is available as a PDF (571.5 KB).

Selected References

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

  1. Atlung T., Nielsen A., Hansen F. G. Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli. J Bacteriol. 1989 Mar;171(3):1683–1691. doi: 10.1128/jb.171.3.1683-1691.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldini M. M., Kaper J. B., Levine M. M., Candy D. C., Moon H. W. Plasmid-mediated adhesion in enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr. 1983;2(3):534–538. doi: 10.1097/00005176-198302030-00023. [DOI] [PubMed] [Google Scholar]
  3. Baldini M. M., Nataro J. P., Kaper J. B. Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli. Infect Immun. 1986 Apr;52(1):334–336. doi: 10.1128/iai.52.1.334-336.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
  6. Caron J., Coffield L. M., Scott J. R. A plasmid-encoded regulatory gene, rns, required for expression of the CS1 and CS2 adhesins of enterotoxigenic Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(3):963–967. doi: 10.1073/pnas.86.3.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donnenberg M. S., Kaper J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310–4317. doi: 10.1128/iai.59.12.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donnenberg M. S., Tacket C. O., James S. P., Losonsky G., Nataro J. P., Wasserman S. S., Kaper J. B., Levine M. M. Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection. J Clin Invest. 1993 Sep;92(3):1412–1417. doi: 10.1172/JCI116717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donnenberg M. S., Yu J., Kaper J. B. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol. 1993 Aug;175(15):4670–4680. doi: 10.1128/jb.175.15.4670-4680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dorman C. J. The VirF protein from Shigella flexneri is a member of the AraC transcription factor superfamily and is highly homologous to Rns, a positive regulator of virulence genes in enterotoxigenic Escherichia coli. Mol Microbiol. 1992 Jun;6(11):1575–1575. doi: 10.1111/j.1365-2958.1992.tb00879.x. [DOI] [PubMed] [Google Scholar]
  12. Débarbouillé M., Raibaud O. Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter. J Bacteriol. 1983 Mar;153(3):1221–1227. doi: 10.1128/jb.153.3.1221-1227.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foubister V., Rosenshine I., Donnenberg M. S., Finlay B. B. The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells. Infect Immun. 1994 Jul;62(7):3038–3040. doi: 10.1128/iai.62.7.3038-3040.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Franke J., Franke S., Schmidt H., Schwarzkopf A., Wieler L. H., Baljer G., Beutin L., Karch H. Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids. J Clin Microbiol. 1994 Oct;32(10):2460–2463. doi: 10.1128/jcm.32.10.2460-2463.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Girón J. A., Ho A. S., Schoolnik G. K. An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science. 1991 Nov 1;254(5032):710–713. doi: 10.1126/science.1683004. [DOI] [PubMed] [Google Scholar]
  16. Jerse A. E., Gicquelais K. G., Kaper J. B. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli. Infect Immun. 1991 Nov;59(11):3869–3875. doi: 10.1128/iai.59.11.3869-3875.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jerse A. E., Kaper J. B. The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid. Infect Immun. 1991 Dec;59(12):4302–4309. doi: 10.1128/iai.59.12.4302-4309.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jerse A. E., Yu J., Tall B. D., Kaper J. B. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7839–7843. doi: 10.1073/pnas.87.20.7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knutton S., Lloyd D. R., McNeish A. S. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987 Jan;55(1):69–77. doi: 10.1128/iai.55.1.69-77.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Knöchel W., Pöting A., Köster M., el Baradi T., Nietfeld W., Bouwmeester T., Pieler T. Evolutionary conserved modules associated with zinc fingers in Xenopus laevis. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6097–6100. doi: 10.1073/pnas.86.16.6097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levine M. M., Edelman R. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: epidemiology and pathogenesis. Epidemiol Rev. 1984;6:31–51. doi: 10.1093/oxfordjournals.epirev.a036274. [DOI] [PubMed] [Google Scholar]
  22. Levine M. M., Nataro J. P., Karch H., Baldini M. M., Kaper J. B., Black R. E., Clements M. L., O'Brien A. D. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J Infect Dis. 1985 Sep;152(3):550–559. doi: 10.1093/infdis/152.3.550. [DOI] [PubMed] [Google Scholar]
  23. Levine M. M., Xu J. G., Kaper J. B., Lior H., Prado V., Tall B., Nataro J., Karch H., Wachsmuth K. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. J Infect Dis. 1987 Jul;156(1):175–182. doi: 10.1093/infdis/156.1.175. [DOI] [PubMed] [Google Scholar]
  24. Lundrigan M. D., Friedrich M. J., Kadner R. J. Nucleotide sequence of the Escherichia coli porin thermoregulatory gene envY. Nucleic Acids Res. 1989 Jan 25;17(2):800–800. doi: 10.1093/nar/17.2.800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maas R. An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid. 1983 Nov;10(3):296–298. doi: 10.1016/0147-619x(83)90045-8. [DOI] [PubMed] [Google Scholar]
  26. McDaniel T. K., Jarvis K. G., Donnenberg M. S., Kaper J. B. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. doi: 10.1073/pnas.92.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  28. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moon H. W., Whipp S. C., Argenzio R. A., Levine M. M., Giannella R. A. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983 Sep;41(3):1340–1351. doi: 10.1128/iai.41.3.1340-1351.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moseley S. L., Huq I., Alim A. R., So M., Samadpour-Motalebi M., Falkow S. Detection of enterotoxigenic Escherichia coli by DNA colony hybridization. J Infect Dis. 1980 Dec;142(6):892–898. doi: 10.1093/infdis/142.6.892. [DOI] [PubMed] [Google Scholar]
  31. Nataro J. P., Maher K. O., Mackie P., Kaper J. B. Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli. Infect Immun. 1987 Oct;55(10):2370–2377. doi: 10.1128/iai.55.10.2370-2377.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nataro J. P., Scaletsky I. C., Kaper J. B., Levine M. M., Trabulsi L. R. Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli. Infect Immun. 1985 May;48(2):378–383. doi: 10.1128/iai.48.2.378-383.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nicholson E. B., Concaugh E. A., Foxall P. A., Island M. D., Mobley H. L. Proteus mirabilis urease: transcriptional regulation by UreR. J Bacteriol. 1993 Jan;175(2):465–473. doi: 10.1128/jb.175.2.465-473.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  35. Priimägi A. F., Mizrokhi L. J., Ilyin Y. V. The Drosophila mobile element jockey belongs to LINEs and contains coding sequences homologous to some retroviral proteins. Gene. 1988 Oct 30;70(2):253–262. doi: 10.1016/0378-1119(88)90197-7. [DOI] [PubMed] [Google Scholar]
  36. Ramos J. L., Rojo F., Zhou L., Timmis K. N. A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators. Nucleic Acids Res. 1990 Apr 25;18(8):2149–2152. doi: 10.1093/nar/18.8.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sakai T., Sasakawa C., Yoshikawa M. Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kiloDalton virF protein. Mol Microbiol. 1988 Sep;2(5):589–597. doi: 10.1111/j.1365-2958.1988.tb00067.x. [DOI] [PubMed] [Google Scholar]
  38. Savelkoul P. H., Willshaw G. A., McConnell M. M., Smith H. R., Hamers A. M., van der Zeijst B. A., Gaastra W. Expression of CFA/I fimbriae is positively regulated. Microb Pathog. 1990 Feb;8(2):91–99. doi: 10.1016/0882-4010(90)90073-y. [DOI] [PubMed] [Google Scholar]
  39. Tobin J. F., Schleif R. F. Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. J Mol Biol. 1987 Aug 20;196(4):789–799. doi: 10.1016/0022-2836(87)90405-0. [DOI] [PubMed] [Google Scholar]
  40. Weeda G., van Ham R. C., Masurel R., Westerveld A., Odijk H., de Wit J., Bootsma D., van der Eb A. J., Hoeijmakers J. H. Molecular cloning and biological characterization of the human excision repair gene ERCC-3. Mol Cell Biol. 1990 Jun;10(6):2570–2581. doi: 10.1128/mcb.10.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES