Summary
Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.
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Andrup L, Atlung T, Ogasawsa N, Yoshikawa H, Hansen FG (1988) Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein. J Bacteriol 170:1333–1338
Bertani G (1951) Studies on lysogenesis. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Del Sal G, Manfioletti G, Schneider C (1989) The CTAB-DNA precipitation method: A common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques 7:514–519
Dykes CW, Halliday IJ, Read MJ, Hobden AN, Harford S (1985) Nucleotide sequences of four variants of porcine enterotoxigenic Escherichia coli. Infect Immun 50:279–283
Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P (1979) Nucleotide sequence of the Hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature 281:646–650
Gaastra W (1984) Enzyme linked immunosorbant assay. In: Walker JM (ed) Methods in molecular biology. Clifton NJ: Humana Press
Gaastra W, Mooi FR, Stuitje AR, de Graaf FK (1981) The nucleotide sequence of the gene encoding the K88ab protein subunit of porcine enterotoxigenic E. coli. FEMS Microbiol Lett 12:41–46
Gaastra W, Klemm P, de Graaf FK (1983) The nucleotide sequence of the K88ad protein subunit of porcine enterotoxigenic E. coli. FEMS Microbiol Lett 18:177–183
Gottesman S (1989) Genetics of proteolysis in Escherichia coli. Annu Rev Genet 23:163–198
Guinee PAM, Jansen WH (1979) Behavior of Escherichia coli K antigens K88ab, K88ac and K88ad in immunoelectrophoresis, double diffusion and hemagglutination. Infect Immun 23:700–705
Jones GW, Rutter JM (1972) Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by Escherichia coli in piglets. Infect Immun 6:918–927
Josephsen J, Hansen F, de Graaf FK, Gaastra W (1984) The nucleotide sequence of the protein subunit of the K88ac fimbria of enterotoxigenic E. coli. FEMS Microbiol Lett 25:301–306
Klemm P (1981) The complete aminoacid sequence of the K88 antigen, a fimbrial protein from E. coli. Eur J Biochem 117:617–627
Kyhse-Andersen J (1984) Electroblotting of multiple gels; a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biophys Biochem Methods 10:203–209
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature 227:680–685
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Mooi FR, de Graaf FK (1979a) Isolation and characterization of the K88 antigen. FEMS Microbiol Lett 5:17–20
Mooi FR, de Graaf FK (1979b) Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen. Nucleic Acids Res 6:849–865
Mooi FR, de Graaf FK (1985) Molecular biology of fimbriae from enterotoxigenic Escherichia coli. Curr Top Microbiol Immunol 118:119–138
Mooi FR, Harms N, Bakker D, de Graaf FK (1981) Organization and expression of genes involved in the production of the K88ab antigen. Infect Immun 32:1155–1163
Mooi FR, Wouters C, Wijfjes A, de Graaf FK (1982) Construction and characterization mutants impaired in the biosynthesis of the K88ab antigen. J Bacteriol 150:512–521
Mooi FR, Wijfjes A, de Graaf FK (1983) Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli. J Bacteriol 154:41–49
Neurath AR, Kent SBH, Strick N (1984) Location and chemical synthesis of a pre-S gene coded immunodominant epitope of Hepatitis B virus. Science 224:392–395
Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium. J Biol Chem 247:3962–3972
Oudega B, de Graaf M, de Boer L, Bakker D, Vader CEM, Mooi FR, de Graaf FK (1989) Detection and identification of FaeC as a minor component of K88 fimbriae of Escherichia coli. Mol Microbiol 3:645–652
Pedersen PA (1991) Structure-function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic E. coli Mol Microbiol 5:1081–1089
Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Van Doorn J, Oudega B, Mooi FR, de Graaf FK (1982) Subcellular localization of polypeptides involved in the biosynthesis of K88ab fimbriae. FEMS Microbiol Lett 13:99–104
Ørskov I, Ørskov F, Leach JM, Sojka J (1961) Simultaneous occurrence of E. coli B and L antigens in strains from diseased swine. Acta Pathol Microbiol Scand [B] 53:404–422
Ørskov I, Orskov F, Sojka WJ, Wittig W (1964) K antigens K88ab(L) and K88ac(L) in Escherichia coli. Acta Pathol Microbiol Scand 62:439–477
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Pedersen, P.A., Andersen, L.N. Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli . Molec. Gen. Genet. 229, 285–291 (1991). https://doi.org/10.1007/BF00272168
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DOI: https://doi.org/10.1007/BF00272168