1887

Abstract

A cluster of 14 genes located on the large plasmid of enteropathogenic (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant host. The fifth gene in the cluster, , encodes a protein that is predicted to be localized to the periplasmic space. To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar mutant EPEC strain by allelic exchange. The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP. These phenotypes were restored to the mutant by a plasmid containing . There was no difference between the wild-type and mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes. Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm. Thus, BfpU represents a novel protein required for type IV pilus assembly.

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2002-08-01
2019-10-19
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References

  1. Alm, R. A. & Mattick, J. S. ( 1995; ). Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence. Mol Microbiol 16, 485-496.[CrossRef]
    [Google Scholar]
  2. Alm, R. A. & Mattick, J. S. ( 1997; ). Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192, 89-98.[CrossRef]
    [Google Scholar]
  3. Alm, R. A., Bodero, A. J., Free, P. D. & Mattick, J. S. ( 1996a; ). Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178, 46-53.
    [Google Scholar]
  4. Alm, R. A., Hallinan, J. P., Watson, A. A. & Mattick, J. S. ( 1996b; ). Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol Microbiol 22, 161-173.[CrossRef]
    [Google Scholar]
  5. Anantha, R. P., Stone, K. D. & Donnenberg, M. S. ( 1998; ). Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells. Infect Immun 66, 122-131.
    [Google Scholar]
  6. Anantha, R. P., Stone, K. D. & Donnenberg, M. S. ( 2000; ). Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili. J Bacteriol 182, 2498-2506.[CrossRef]
    [Google Scholar]
  7. Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H. & Tommassen, J. ( 1998; ). Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27, 209-219.[CrossRef]
    [Google Scholar]
  8. Blank, T. E. & Donnenberg, M. S. ( 2001; ). Novel topology of BfpE, a cytoplasmic membrane protein required for type IV fimbrial biogenesis in enteropathogenic Escherichia coli. J Bacteriol 183, 4435-4450.[CrossRef]
    [Google Scholar]
  9. Blank, T. E., Zhong, H., Bell, A. L., Whittam, T. S. & Donnenberg, M. S. ( 2000; ). Molecular variation among type IV pilin (bfpA) genes from diverse enteropathogenic Escherichia coli strains. Infect Immun 68, 7028-7038.[CrossRef]
    [Google Scholar]
  10. Castric, P. ( 1995; ). pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin. Microbiology 141, 1247-1254.[CrossRef]
    [Google Scholar]
  11. Donnenberg, M. S. & Kaper, J. B. ( 1991; ). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59, 4310-4317.
    [Google Scholar]
  12. Donnenberg, M. S. & Nataro, J. P. ( 1995; ). Methods for studying adhesion of diarrheagenic Escherichia coli. Methods Enzymol 253, 324-336.
    [Google Scholar]
  13. Donnenberg, M. S., Girón, J. A., Nataro, J. P. & Kaper, J. B. ( 1992; ). A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol Microbiol 6, 3427-3437.[CrossRef]
    [Google Scholar]
  14. Donnenberg, M. S., Yu, J. & Kaper, J. B. ( 1993; ). A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol 175, 4670-4680.
    [Google Scholar]
  15. Forest, K. T., Dunham, S. A., Koomey, M. & Tainer, J. A. ( 1999; ). Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology. Mol Microbiol 31, 743-752.[CrossRef]
    [Google Scholar]
  16. Fussenegger, M., Rudel, T., Barten, R., Ryll, R. & Meyer, T. F. ( 1997; ). Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae – a review. Gene 192, 125-134.[CrossRef]
    [Google Scholar]
  17. Gerberding, H. & Mayer, F. ( 1988; ). Localization of the membrane-bound hydrogenase in Alcaligenes eutrophus by electron microscopic immunocytochemistry. FEMS Microbiol Lett 50, 265-270.[CrossRef]
    [Google Scholar]
  18. Girón, J. A., Qadri, F., Azim, T., Jarvis, K. J., Kaper, J. B. & Albert, M. J. ( 1995; ). Monoclonal antibodies specific for the bundle-forming pilus of enteropathogenic Escherichia coli. Infect Immun 63, 4949-4952.
    [Google Scholar]
  19. Hobbs, M. & Mattick, J. S. ( 1993; ). Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol Microbiol 10, 233-243.[CrossRef]
    [Google Scholar]
  20. Iredell, J. R. & Manning, P. A. ( 1994; ). The toxin-co-regulated pilus of Vibrio cholerae O1: a model for type 4 pilus biogenesis? Trends Microbiol 2, 187-192.[CrossRef]
    [Google Scholar]
  21. Jacob-Dubuisson, F., Pinkner, J., Xu, Z., Striker, R., Padmanhaban, A. & Hultgren, S. J. ( 1994; ). PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA. Proc Natl Acad Sci USA 91, 11552-11556.[CrossRef]
    [Google Scholar]
  22. Jerse, A. E. & Kaper, J. B. ( 1991; ). 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 59, 4302-4309.
    [Google Scholar]
  23. Kim, S. R. & Komano, T. ( 1997; ). The plasmid R64 thin pilus identified as a type IV pilus. J Bacteriol 179, 3594-3603.
    [Google Scholar]
  24. LaPointe, C. F. & Taylor, R. K. ( 2000; ). The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases. J Biol Chem 275, 1502-1510.[CrossRef]
    [Google Scholar]
  25. Levine, M. M., Bergquist, E. J., Nalin, D. R., Waterman, D. H., Hornick, R. B., Young, C. R., Sotman, S. & Rowe, B. ( 1978; ). Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1, 1119-1122.
    [Google Scholar]
  26. Levine, M. M., Nataro, J. P., Karch, H., Baldini, M. M., Kaper, J. B., Black, R. E., Clements, M. L. & O’Brien, A. D. ( 1985; ). 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 152, 550-559.[CrossRef]
    [Google Scholar]
  27. Lindberg, F., Tennent, J. M., Hultgren, S. J., Lund, B. & Normark, S. ( 1989; ). PapD, a periplasmic transport protein in P-pilus biogenesis. J Bacteriol 171, 6052-6058.
    [Google Scholar]
  28. McNamara, B. P. & Donnenberg, M. S. ( 2000; ). Evidence for specificity in type 4 pilus biogenesis by enteropathogenic Escherichia coli. Microbiology 146, 719-729.
    [Google Scholar]
  29. Ménard, R., Sansonetti, P. J. & Parsot, C. ( 1993; ). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899-5906.
    [Google Scholar]
  30. Merz, A. J., So, M. & Sheetz, M. P. ( 2000; ). Pilus retraction powers bacterial twitching motility. Nature 407, 98-102.[CrossRef]
    [Google Scholar]
  31. Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A., Ghazi, A. & Pugsley, A. P. ( 1999; ). Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci USA 96, 8173-8177.[CrossRef]
    [Google Scholar]
  32. Nouwen, N., Stahlberg, H., Pugsley, A. P. & Engel, A. ( 2000; ). Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. EMBO J 19, 2229-2236.[CrossRef]
    [Google Scholar]
  33. Nunn, D., Bergman, S. & Lory, S. ( 1990; ). Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172, 2911-2919.
    [Google Scholar]
  34. Paranchych, W. & Frost, L. S. ( 1988; ). The physiology and biochemistry of pili. Adv Microb Physiol 29, 53-114.
    [Google Scholar]
  35. Parge, H. E., Forest, K. T., Hickey, M. J., Christensen, D. A., Getzoff, E. D. & Tainer, J. A. ( 1995; ). Structure of the fibre-forming protein pilin at 2·6 Å resolution. Nature 378, 32-38.[CrossRef]
    [Google Scholar]
  36. Pugsley, A. P. ( 1993; ). The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57, 50-108.
    [Google Scholar]
  37. Roth, J., Bendayan, M., Carlemalm, E., Villiger, W. & Garavito, M. ( 1981; ). Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29, 663-671.[CrossRef]
    [Google Scholar]
  38. Russel, M. ( 1998; ). Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J Mol Biol 279, 485-499.[CrossRef]
    [Google Scholar]
  39. Schmidt, S. A., Bieber, D., Ramer, S. W., Hwang, J., Wu, C. Y. & Schoolnik, G. ( 2001; ). Structure–function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli. J Bacteriol 183, 4848-4859.[CrossRef]
    [Google Scholar]
  40. Sohel, I., Puente, J. L., Ramer, S. W., Bieber, D., Wu, C.-Y. & Schoolnik, G. K. ( 1996; ). Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis. J Bacteriol 178, 2613-2628.
    [Google Scholar]
  41. Spangenberg, C., Fislage, R., Römling, U. & Tümmler, B. ( 1997; ). Disrespectful type IV pilins. Mol Microbiol 25, 203-204.
    [Google Scholar]
  42. Stimson, E., Virji, M., Makepeace, K. & 9 other authors ( 1995; ). Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose. Mol Microbiol 17, 1201–1214.[CrossRef]
    [Google Scholar]
  43. Stone, K. D., Zhang, H.-Z., Carlson, L. K. & Donnenberg, M. S. ( 1996; ). A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for biogenesis of a type IV pilus. Mol Microbiol 20, 325-337.[CrossRef]
    [Google Scholar]
  44. Strom, M. S., Nunn, D. N. & Lory, S. ( 1993; ). A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc Natl Acad Sci USA 90, 2404-2408.[CrossRef]
    [Google Scholar]
  45. Taniguchi, T., Akeda, Y., Haba, A., Yasuda, Y., Yamamoto, K., Honda, T. & Tochikubo, K. ( 2001; ). Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli. Infect Immun 69, 5864-5873.[CrossRef]
    [Google Scholar]
  46. Thorstenson, Y. R., Zhang, Y., Olson, P. S. & Mascarenhas, D. ( 1997; ). Leaderless polypeptides efficiently extracted from whole cells by osmotic shock. J Bacteriol 179, 5333-5339.
    [Google Scholar]
  47. Tobe, T., Schoolnik, G. K., Sohel, I., Bustamante, V. H. & Puente, J. L. ( 1996; ). Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli. Mol Microbiol 21, 963-975.[CrossRef]
    [Google Scholar]
  48. Towbin, H., Staehelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354.[CrossRef]
    [Google Scholar]
  49. Virji, M., Saunders, J. R., Sims, G., Makepeace, K., Maskell, D. & Ferguson, D. J. P. ( 1993; ). Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin. Mol Microbiol 10, 1013-1028.[CrossRef]
    [Google Scholar]
  50. Wang, R. F. & Kushner, S. R. ( 1991; ). Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100, 195-199.[CrossRef]
    [Google Scholar]
  51. Wolfgang, M., Lauer, P., Park, H. S., Brossay, L., Hébert, J. & Koomey, M. ( 1998; ). PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 29, 321-330.[CrossRef]
    [Google Scholar]
  52. Wolfgang, M., van Putten, J. P., Hayes, S. F., Dorward, D. & Koomey, M. ( 2000; ). Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19, 6408-6418.[CrossRef]
    [Google Scholar]
  53. Yoshida, T., Kim, S. R. & Komano, T. ( 1999; ). Twelve pil genes are required for biogenesis of the R64 thin pilus. J Bacteriol 181, 2038-2043.
    [Google Scholar]
  54. Zhang, H.-Z. & Donnenberg, M. S. ( 1996; ). DsbA is required for stability of the type IV pilin of enteropathogenic Escherichia coli. Mol Microbiol 21, 787-797.[CrossRef]
    [Google Scholar]
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