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. 1988 Aug;170(8):3350–3358. doi: 10.1128/jb.170.8.3350-3358.1988

Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

M S Hanson 1, J Hempel 1, C C Brinton Jr 1
PMCID: PMC211301  PMID: 2900235

Abstract

Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1.

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Selected References

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  1. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BRINTON C. C., Jr, BUZZELL A., LAUFFER M. A. Electrophoresis and phage susceptibility studies on a filament-producing variant of the E. coli B bacterium. Biochim Biophys Acta. 1954 Dec;15(4):533–542. doi: 10.1016/0006-3002(54)90011-6. [DOI] [PubMed] [Google Scholar]
  3. BRINTON C. C., Jr, GEMSKI P., Jr, CARNAHAN J. A NEW TYPE OF BACTERIAL PILUS GENETICALLY CONTROLLED BY THE FERTILITY FACTOR OF E. COLI K 12 AND ITS ROLE IN CHROMOSOME TRANSFER. Proc Natl Acad Sci U S A. 1964 Sep;52:776–783. doi: 10.1073/pnas.52.3.776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BRINTON C. C., Jr Non-flagellar appendages of bacteria. Nature. 1959 Mar 21;183(4664):782–786. doi: 10.1038/183782a0. [DOI] [PubMed] [Google Scholar]
  5. Brinton C. C., Jr The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans N Y Acad Sci. 1965 Jun;27(8):1003–1054. doi: 10.1111/j.2164-0947.1965.tb02342.x. [DOI] [PubMed] [Google Scholar]
  6. Båga M., Norgren M., Normark S. Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation. Cell. 1987 Apr 24;49(2):241–251. doi: 10.1016/0092-8674(87)90565-4. [DOI] [PubMed] [Google Scholar]
  7. Cheney C. P., Formal S. B., Schad P. A., Boedeker E. C. Genetic transfer of a mucosal adherence factor (R1) from an enteropathogenic Escherichia coli strain into a Shigella flexneri strain and the phenotypic suppression of this adherence factor. J Infect Dis. 1983 Apr;147(4):711–723. doi: 10.1093/infdis/147.4.711. [DOI] [PubMed] [Google Scholar]
  8. ENGLANDER S. W., EPSTEIN H. T. Optical methods for measuring nucleoprotein and nucleic acid concentrations. Arch Biochem Biophys. 1957 May;68(1):144–149. doi: 10.1016/0003-9861(57)90334-x. [DOI] [PubMed] [Google Scholar]
  9. Eisenstein B. I. Phase variation of type 1 fimbriae in Escherichia coli is under transcriptional control. Science. 1981 Oct 16;214(4518):337–339. doi: 10.1126/science.6116279. [DOI] [PubMed] [Google Scholar]
  10. Eshdat Y., Silverblatt F. J., Sharon N. Dissociation and reassembly of Escherichia coli type 1 pili. J Bacteriol. 1981 Oct;148(1):308–314. doi: 10.1128/jb.148.1.308-314.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaastra W., de Graaf F. K. Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol Rev. 1982 Jun;46(2):129–161. doi: 10.1128/mr.46.2.129-161.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hanson M. S., Brinton C. C., Jr Identification and characterization of E. coli type-1 pilus tip adhesion protein. Nature. 1988 Mar 17;332(6161):265–268. doi: 10.1038/332265a0. [DOI] [PubMed] [Google Scholar]
  13. Hawke D., Yuan P. M., Shively J. E. Microsequence analysis of peptides and proteins. II. Separation of amino acid phenylthiohydantoin derivatives by high-performance liquid chromatography on octadecylsilane supports. Anal Biochem. 1982 Mar 1;120(2):302–311. doi: 10.1016/0003-2697(82)90351-7. [DOI] [PubMed] [Google Scholar]
  14. Hayashi K. A rapid determination of sodium dodecyl sulfate with methylene blue. Anal Biochem. 1975 Aug;67(2):503–506. doi: 10.1016/0003-2697(75)90324-3. [DOI] [PubMed] [Google Scholar]
  15. Henderson L. E., Oroszlan S., Konigsberg W. A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction. Anal Biochem. 1979 Feb;93(1):153–157. [PubMed] [Google Scholar]
  16. Hermodson M. A., Chen K. C., Buchanan T. M. Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid. Biochemistry. 1978 Feb 7;17(3):442–445. doi: 10.1021/bi00596a010. [DOI] [PubMed] [Google Scholar]
  17. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  18. Jörnvall H., Philipson L. Limited proteolysis and a reactive cysteine residue define accesible regions in the native conformation of the adenovirus hexon protein. Eur J Biochem. 1980 Feb;104(1):237–247. doi: 10.1111/j.1432-1033.1980.tb04421.x. [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. Klemm P. Fimbrial adhesions of Escherichia coli. Rev Infect Dis. 1985 May-Jun;7(3):321–340. doi: 10.1093/clinids/7.3.321. [DOI] [PubMed] [Google Scholar]
  21. Klemm P. The complete amino-acid sequence of the K88 antigen, a fimbrial protein from Escherichia coli. Eur J Biochem. 1981 Jul;117(3):617–627. doi: 10.1111/j.1432-1033.1981.tb06382.x. [DOI] [PubMed] [Google Scholar]
  22. Korhonen T. K., Nurmiaho E. L., Ranta H., Edén C. S. New Method for isolation of immunologically pure pili from Escherichia coli. Infect Immun. 1980 Feb;27(2):569–575. doi: 10.1128/iai.27.2.569-575.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Korhonen T. K., Väisänen-Rhen V., Rhen M., Pere A., Parkkinen J., Finne J. Escherichia coli fimbriae recognizing sialyl galactosides. J Bacteriol. 1984 Aug;159(2):762–766. doi: 10.1128/jb.159.2.762-766.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Labigne-Roussel A. F., Lark D., Schoolnik G., Falkow S. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect Immun. 1984 Oct;46(1):251–259. doi: 10.1128/iai.46.1.251-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Levitt M. A simplified representation of protein conformations for rapid simulation of protein folding. J Mol Biol. 1976 Jun 14;104(1):59–107. doi: 10.1016/0022-2836(76)90004-8. [DOI] [PubMed] [Google Scholar]
  28. Maurer L., Orndorff P. E. Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili. J Bacteriol. 1987 Feb;169(2):640–645. doi: 10.1128/jb.169.2.640-645.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyer T. F., Mlawer N., So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell. 1982 Aug;30(1):45–52. doi: 10.1016/0092-8674(82)90010-1. [DOI] [PubMed] [Google Scholar]
  30. Morgan R. L., Isaacson R. E., Moon H. W., Brinton C. C., To C. C. Immunization of suckling pigs against enterotoxigenic Escherichia coli-induced diarrheal disease by vaccinating dams with purified 987 or K99 pili: protection correlates with pilus homology of vaccine and challenge. Infect Immun. 1978 Dec;22(3):771–777. doi: 10.1128/iai.22.3.771-777.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  32. Nagy B., Moon H. W., Isaacson R. E., To C. C., Brinton C. C. Immunization of suckling pigs against enteric enterotoxigenic Escherichia coli infection by vaccinating dams with purified pili. Infect Immun. 1978 Jul;21(1):269–274. doi: 10.1128/iai.21.1.269-274.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Old D. C. Inhibition of the interaction between fimbrial haemagglutinins and erythrocytes by D-mannose and other carbohydrates. J Gen Microbiol. 1972 Jun;71(1):149–157. doi: 10.1099/00221287-71-1-149. [DOI] [PubMed] [Google Scholar]
  34. Orndorff P. E., Falkow S. Nucleotide sequence of pilA, the gene encoding the structural component of type 1 pili in Escherichia coli. J Bacteriol. 1985 Apr;162(1):454–457. doi: 10.1128/jb.162.1.454-457.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rhen M., Klemm P., Korhonen T. K. Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in Escherichia coli K-12. J Bacteriol. 1986 Dec;168(3):1234–1242. doi: 10.1128/jb.168.3.1234-1242.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Salit I. E., Gotschlich E. C. Type I Escherichia coli pili: characterization of binding to monkey kidney cells. J Exp Med. 1977 Nov 1;146(5):1182–1194. doi: 10.1084/jem.146.5.1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  38. Swaney L. M., Liu Y. P., Ippen-Ihler K., Brinton C. C., Jr Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants. J Bacteriol. 1977 Apr;130(1):506–511. doi: 10.1128/jb.130.1.506-511.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swaney L. M., Liu Y. P., To C. M., To C. C., Ippen-Ihler K., Brinton C. C., Jr Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production. J Bacteriol. 1977 Apr;130(1):495–505. doi: 10.1128/jb.130.1.495-505.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Väisänen-Rhen V. Fimbria-like hemagglutinin of Escherichia coli O75 strains. Infect Immun. 1984 Nov;46(2):401–407. doi: 10.1128/iai.46.2.401-407.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  42. de Graaf F. K., Roorda I. Production, purification, and characterization of the fimbrial adhesive antigen F41 isolated from calf enteropathogenic Escherichia coli strain B41M. Infect Immun. 1982 May;36(2):751–758. doi: 10.1128/iai.36.2.751-758.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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