Abstract
The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, site-specific, mutagenesis. The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively. The threonine-133 mutant fibrillae were indistinguishable from wild-type fibrillae. In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules. After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit.
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- Beachey E. H. Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surface. J Infect Dis. 1981 Mar;143(3):325–345. doi: 10.1093/infdis/143.3.325. [DOI] [PubMed] [Google Scholar]
- 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]
- Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
- 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]
- Guinée P. A., Jansen W. H., Agterberg C. M. Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with entertoxigenicity. Infect Immun. 1976 May;13(5):1369–1377. doi: 10.1128/iai.13.5.1369-1377.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hacker J., Schmidt G., Hughes C., Knapp S., Marget M., Goebel W. Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain. Infect Immun. 1985 Feb;47(2):434–440. doi: 10.1128/iai.47.2.434-440.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacobs A. A., van Mechelen J. R., de Graaf F. K. Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli. Biochim Biophys Acta. 1985 Nov 29;832(2):148–155. doi: 10.1016/0167-4838(85)90326-7. [DOI] [PubMed] [Google Scholar]
- Jacobs A. A., van den Berg P. A., Bak H. J., de Graaf F. K. Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli. Biochim Biophys Acta. 1986 Jul 25;872(1-2):92–97. doi: 10.1016/0167-4838(86)90151-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Lindberg F. P., Lund B., Normark S. Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells. EMBO J. 1984 May;3(5):1167–1173. doi: 10.1002/j.1460-2075.1984.tb01946.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindberg F., Lund B., Normark S. Gene products specifying adhesion of uropathogenic Escherichia coli are minor components of pili. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1891–1895. doi: 10.1073/pnas.83.6.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ludwig D. S., Holmes R. K., Schoolnik G. K. Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit. J Biol Chem. 1985 Oct 15;260(23):12528–12534. [PubMed] [Google Scholar]
- Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooi F. R., de Graaf F. K., van Embden J. D. Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen. Nucleic Acids Res. 1979 Mar;6(3):849–865. doi: 10.1093/nar/6.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooi F. R., van Buuren M., Koopman G., Roosendaal B., de Graaf F. K. K88ab gene of Escherichia coli encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin. J Bacteriol. 1984 Aug;159(2):482–487. doi: 10.1128/jb.159.2.482-487.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norgren M., Normark S., Lark D., O'Hanley P., Schoolnik G., Falkow S., Svanborg-Edén C., Båga M., Uhlin B. E. Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation. EMBO J. 1984 May;3(5):1159–1165. doi: 10.1002/j.1460-2075.1984.tb01945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riordan J. F. Functional arginyl residues in carboxypeptidase A. Modification with butanedione. Biochemistry. 1973 Sep 25;12(20):3915–3923. doi: 10.1021/bi00744a020. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smit H., Gaastra W., Kamerling J. P., Vliegenthart J. F., de Graaf F. K. Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin. Infect Immun. 1984 Nov;46(2):578–584. doi: 10.1128/iai.46.2.578-584.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]