Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Aug;176(15):4691–4699. doi: 10.1128/jb.176.15.4691-4699.1994

AggR, a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli.

J P Nataro 1, D Yikang 1, D Yingkang 1, K Walker 1
PMCID: PMC196291  PMID: 7913930

Abstract

Enteroaggregative Escherichia coli (EAggEC) has been associated with persistent pediatric diarrhea in the developing world, yet the pathogenetic mechanisms of EAggEC infection are unknown. Our previous data have suggested that aggregative adherence of some EAggEC strains to HEp-2 cells is mediated by flexible, bundle-forming fimbriae, which we have termed aggregative adherence fimbriae I (AAF/I). Genes sufficient to confer expression of AAF/I are located on the 60-MDa plasmid of EAggEC 17-2; AAF/I genes are present as two unlinked plasmid regions (regions 1 and 2), separated by 9 kb of DNA. Here we report the complete DNA sequencing of region 2 and the identification of an open reading frame which is involved in the expression of AAF/I. One open reading frame of 794 bp encodes a protein (designated AggR) with a predicted molecular size of 29.4 kDa, which shows a high degree of amino acid sequence identity to CfaR and other members of the AraC class of gene regulators. The cloned aggR gene (or, alternatively, a cloned cfaR gene) was sufficient to complement a region 1 clone to confer AAF/I expression. To further substantiate the role of aggR in the regulation of AAF/I, we constructed a 289-bp in-frame aggR deletion and replaced the native gene in 17-2 by allelic exchange, using the temperature-sensitive vector pIB307. The resulting aggR deletions were negative for AAF/I expression, but expression was restored when the aggR gene (cloned into pBluescript II SK) was reintroduced into the aggR mutant. RNA slot blot experiments using a probe for the putative AAF/I pilin subunit (aggA) revealed that aggR operates as a transcriptional activator of aggA expression. aggA::phoA fusions were constructed in 17-2 and in 17-2 delta aggR. AggR was found to promote expression of the aggA gene under a variety of conditions of temperature, osmolarity, oxygen tension, and medium. At acid pH, aggA expression was maximal and was regulated by both AggR-dependent and AggR-independent mechanisms.

Full text

PDF
4691

Images in this article

4696Image
on p.4696

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. Bhan M. K., Khoshoo V., Sommerfelt H., Raj P., Sazawal S., Srivastava R. Enteroaggregative Escherichia coli and Salmonella associated with nondysenteric persistent diarrhea. Pediatr Infect Dis J. 1989 Aug;8(8):499–502. doi: 10.1097/00006454-198908000-00005. [DOI] [PubMed] [Google Scholar]
  3. Bhan M. K., Raj P., Levine M. M., Kaper J. B., Bhandari N., Srivastava R., Kumar R., Sazawal S. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis. 1989 Jun;159(6):1061–1064. doi: 10.1093/infdis/159.6.1061. [DOI] [PubMed] [Google Scholar]
  4. Blomfield I. C., McClain M. S., Eisenstein B. I. Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants. Mol Microbiol. 1991 Jun;5(6):1439–1445. doi: 10.1111/j.1365-2958.1991.tb00790.x. [DOI] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bustos S. A., Schleif R. F. Functional domains of the AraC protein. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5638–5642. doi: 10.1073/pnas.90.12.5638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Caron J., Maneval D. R., Kaper J. B., Scott J. R. Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates. Infect Immun. 1990 Oct;58(10):3442–3444. doi: 10.1128/iai.58.10.3442-3444.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caron J., Scott J. R. A rns-like regulatory gene for colonization factor antigen I (CFA/I) that controls expression of CFA/I pilin. Infect Immun. 1990 Apr;58(4):874–878. doi: 10.1128/iai.58.4.874-878.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cravioto A., Tello A., Navarro A., Ruiz J., Villafán H., Uribe F., Eslava C. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet. 1991 Feb 2;337(8736):262–264. doi: 10.1016/0140-6736(91)90868-p. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Finlay B. B., Falkow S. Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J Infect Dis. 1990 Nov;162(5):1096–1106. doi: 10.1093/infdis/162.5.1096. [DOI] [PubMed] [Google Scholar]
  14. Gallegos M. T., Michán C., Ramos J. L. The XylS/AraC family of regulators. Nucleic Acids Res. 1993 Feb 25;21(4):807–810. doi: 10.1093/nar/21.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  16. Hibberd M. L., McConnell M. M., Willshaw G. A., Smith H. R., Rowe B. Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166. J Gen Microbiol. 1991 Aug;137(8):1963–1970. doi: 10.1099/00221287-137-8-1963. [DOI] [PubMed] [Google Scholar]
  17. Jordi B. J., Dagberg B., de Haan L. A., Hamers A. M., van der Zeijst B. A., Gaastra W., Uhlin B. E. The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli. EMBO J. 1992 Jul;11(7):2627–2632. doi: 10.1002/j.1460-2075.1992.tb05328.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kato J., Ito K., Nakamura A., Watanabe H. Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes. Infect Immun. 1989 May;57(5):1391–1398. doi: 10.1128/iai.57.5.1391-1398.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  20. Levine M. M., Ristaino P., Marley G., Smyth C., Knutton S., Boedeker E., Black R., Young C., Clements M. L., Cheney C. Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun. 1984 May;44(2):409–420. doi: 10.1128/iai.44.2.409-420.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nataro J. P., Deng Y., Maneval D. R., German A. L., Martin W. C., Levine M. M. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect Immun. 1992 Jun;60(6):2297–2304. doi: 10.1128/iai.60.6.2297-2304.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nataro J. P., Kaper J. B., Robins-Browne R., Prado V., Vial P., Levine M. M. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J. 1987 Sep;6(9):829–831. doi: 10.1097/00006454-198709000-00008. [DOI] [PubMed] [Google Scholar]
  25. Nataro J. P., Yikang D., Giron J. A., Savarino S. J., Kothary M. H., Hall R. Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unlinked plasmid regions. Infect Immun. 1993 Mar;61(3):1126–1131. doi: 10.1128/iai.61.3.1126-1131.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ogierman M. A., Manning P. A. Homology of TcpN, a putative regulatory protein of Vibrio cholerae, to the AraC family of transcriptional activators. Gene. 1992 Jul 1;116(1):93–97. doi: 10.1016/0378-1119(92)90634-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Savarino S. J., Fasano A., Watson J., Martin B. M., Levine M. M., Guandalini S., Guerry P. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3093–3097. doi: 10.1073/pnas.90.7.3093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Vial P. A., Mathewson J. J., DuPont H. L., Guers L., Levine M. M. Comparison of two assay methods for patterns of adherence to HEp-2 cells of Escherichia coli from patients with diarrhea. J Clin Microbiol. 1990 May;28(5):882–885. doi: 10.1128/jcm.28.5.882-885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yamamoto T., Endo S., Yokota T., Echeverria P. Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa. Infect Immun. 1991 Oct;59(10):3722–3739. doi: 10.1128/iai.59.10.3722-3739.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Haan L. A., Willshaw G. A., van der Zeijst B. A., Gaastra W. The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5. FEMS Microbiol Lett. 1991 Oct 15;67(3):341–346. doi: 10.1111/j.1574-6968.1991.tb04487.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES