Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.
The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000008:administration & dosage / Q000235:genetics* / Q000302:isolation & purification; D000954:Antigens, Surface / Q000008:administration & dosage / Q000235:genetics* / Q000302:isolation & purification; D001422:Bacterial Adhesion; D001427:Bacterial Toxins; D001428:Bacterial Vaccines / Q000592:standards; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D010861:Fimbriae, Bacterial / Q000276:immunology; D005798:Genes, Bacterial; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014364:Tryptophan / Q000235:genetics* / Q000502:physiology
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; C031429:K99 antigen; D014364:Tryptophan