Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Apr 15;17(8):2177–2185. doi: 10.1093/emboj/17.8.2177

Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.

E T Saulino 1, D G Thanassi 1, J S Pinkner 1, S J Hultgren 1
PMCID: PMC1170562  PMID: 9545231

Abstract

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.

Full Text

The Full Text of this article is available as a PDF (402.3 KB).

Selected References

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

  1. Bitter W., Koster M., Latijnhouwers M., de Cock H., Tommassen J. Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol. 1998 Jan;27(1):209–219. doi: 10.1046/j.1365-2958.1998.00677.x. [DOI] [PubMed] [Google Scholar]
  2. Bullitt E., Jones C. H., Striker R., Soto G., Jacob-Dubuisson F., Pinkner J., Wick M. J., Makowski L., Hultgren S. J. Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12890–12895. doi: 10.1073/pnas.93.23.12890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Connell I., Agace W., Klemm P., Schembri M., Mărild S., Svanborg C. Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9827–9832. doi: 10.1073/pnas.93.18.9827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dodson K. W., Jacob-Dubuisson F., Striker R. T., Hultgren S. J. Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3670–3674. doi: 10.1073/pnas.90.8.3670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fägerstam L. G., Frostell-Karlsson A., Karlsson R., Persson B., Rönnberg I. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis. J Chromatogr. 1992 Apr 24;597(1-2):397–410. doi: 10.1016/0021-9673(92)80137-j. [DOI] [PubMed] [Google Scholar]
  6. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  7. Holmgren A., Bränden C. I. Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. Nature. 1989 Nov 16;342(6247):248–251. doi: 10.1038/342248a0. [DOI] [PubMed] [Google Scholar]
  8. Hull R. A., Gill R. E., Hsu P., Minshew B. H., Falkow S. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun. 1981 Sep;33(3):933–938. doi: 10.1128/iai.33.3.933-938.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hultgren S. J., Schwan W. R., Schaeffer A. J., Duncan J. L. Regulation of production of type 1 pili among urinary tract isolates of Escherichia coli. Infect Immun. 1986 Dec;54(3):613–620. doi: 10.1128/iai.54.3.613-620.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hung D. L., Knight S. D., Woods R. M., Pinkner J. S., Hultgren S. J. Molecular basis of two subfamilies of immunoglobulin-like chaperones. EMBO J. 1996 Aug 1;15(15):3792–3805. [PMC free article] [PubMed] [Google Scholar]
  11. Jacob-Dubuisson F., Striker R., Hultgren S. J. Chaperone-assisted self-assembly of pili independent of cellular energy. J Biol Chem. 1994 Apr 29;269(17):12447–12455. [PubMed] [Google Scholar]
  12. Johnsson B., Löfås S., Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem. 1991 Nov 1;198(2):268–277. doi: 10.1016/0003-2697(91)90424-r. [DOI] [PubMed] [Google Scholar]
  13. Jones C. H., Danese P. N., Pinkner J. S., Silhavy T. J., Hultgren S. J. The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems. EMBO J. 1997 Nov 3;16(21):6394–6406. doi: 10.1093/emboj/16.21.6394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones C. H., Pinkner J. S., Nicholes A. V., Slonim L. N., Abraham S. N., Hultgren S. J. FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8397–8401. doi: 10.1073/pnas.90.18.8397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones C. H., Pinkner J. S., Roth R., Heuser J., Nicholes A. V., Abraham S. N., Hultgren S. J. FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2081–2085. doi: 10.1073/pnas.92.6.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karlsson R., Michaelsson A., Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991 Dec 15;145(1-2):229–240. doi: 10.1016/0022-1759(91)90331-9. [DOI] [PubMed] [Google Scholar]
  17. Kazmierczak B. I., Mielke D. L., Russel M., Model P. pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J Mol Biol. 1994 Apr 29;238(2):187–198. doi: 10.1006/jmbi.1994.1280. [DOI] [PubMed] [Google Scholar]
  18. Klemm P., Christiansen G. The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae. Mol Gen Genet. 1990 Jan;220(2):334–338. doi: 10.1007/BF00260505. [DOI] [PubMed] [Google Scholar]
  19. Klemm P., Christiansen G. Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae. Mol Gen Genet. 1987 Jul;208(3):439–445. doi: 10.1007/BF00328136. [DOI] [PubMed] [Google Scholar]
  20. Krogfelt K. A., Bergmans H., Klemm P. Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae. Infect Immun. 1990 Jun;58(6):1995–1998. doi: 10.1128/iai.58.6.1995-1998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuehn M. J., Ogg D. J., Kihlberg J., Slonim L. N., Flemmer K., Bergfors T., Hultgren S. J. Structural basis of pilus subunit recognition by the PapD chaperone. Science. 1993 Nov 19;262(5137):1234–1241. doi: 10.1126/science.7901913. [DOI] [PubMed] [Google Scholar]
  22. Langermann S., Palaszynski S., Barnhart M., Auguste G., Pinkner J. S., Burlein J., Barren P., Koenig S., Leath S., Jones C. H. Prevention of mucosal Escherichia coli infection by FimH-adhesin-based systemic vaccination. Science. 1997 Apr 25;276(5312):607–611. doi: 10.1126/science.276.5312.607. [DOI] [PubMed] [Google Scholar]
  23. Lee C. A. Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells? Trends Microbiol. 1997 Apr;5(4):148–156. doi: 10.1016/S0966-842X(97)01029-9. [DOI] [PubMed] [Google Scholar]
  24. Linderoth N. A., Simon M. N., Russel M. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science. 1997 Nov 28;278(5343):1635–1638. doi: 10.1126/science.278.5343.1635. [DOI] [PubMed] [Google Scholar]
  25. Minion F. C., Abraham S. N., Beachey E. H., Goguen J. D. The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit. J Bacteriol. 1986 Mar;165(3):1033–1036. doi: 10.1128/jb.165.3.1033-1036.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mitsui Y., Dyer F. P., Langridge R. X-ray diffraction studies of bacterial pili. J Mol Biol. 1973 Sep 5;79(1):57–64. doi: 10.1016/0022-2836(73)90269-6. [DOI] [PubMed] [Google Scholar]
  27. Nikaido H. Isolation of outer membranes. Methods Enzymol. 1994;235:225–234. doi: 10.1016/0076-6879(94)35143-0. [DOI] [PubMed] [Google Scholar]
  28. Orndorff P. E., Falkow S. Organization and expression of genes responsible for type 1 piliation in Escherichia coli. J Bacteriol. 1984 Aug;159(2):736–744. doi: 10.1128/jb.159.2.736-744.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts J. A., Marklund B. I., Ilver D., Haslam D., Kaack M. B., Baskin G., Louis M., Möllby R., Winberg J., Normark S. The Gal(alpha 1-4)Gal-specific tip adhesin of Escherichia coli P-fimbriae is needed for pyelonephritis to occur in the normal urinary tract. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11889–11893. doi: 10.1073/pnas.91.25.11889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Russel M. Moving through the membrane with filamentous phages. Trends Microbiol. 1995 Jun;3(6):223–228. doi: 10.1016/s0966-842x(00)88929-5. [DOI] [PubMed] [Google Scholar]
  31. Russell P. W., Orndorff P. E. Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding. J Bacteriol. 1992 Sep;174(18):5923–5935. doi: 10.1128/jb.174.18.5923-5935.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Salmond G. P. Pili, peptidases and protein secretion: curious connections. Trends Microbiol. 1996 Dec;4(12):474–476. doi: 10.1016/s0966-842x(97)82907-1. [DOI] [PubMed] [Google Scholar]
  33. Thanassi D. G., Saulino E. T., Lombardo M. J., Roth R., Heuser J., Hultgren S. J. The PapC usher forms an oligomeric channel: implications for pilus biogenesis across the outer membrane. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3146–3151. doi: 10.1073/pnas.95.6.3146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Valent Q. A., Zaal J., de Graaf F. K., Oudega B. Subcellular localization and topology of the K88 usher FaeD in Escherichia coli. Mol Microbiol. 1995 Jun;16(6):1243–1257. doi: 10.1111/j.1365-2958.1995.tb02346.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES