Organization and expression of genes involved in the biosynthesis of K99 fimbriae

Abstract

Escherichia coli K-12 minicells were used to study the expression of plasmid pFK99 encoding for the production of K99 fimbriae. Plasmid pFK99 is composed of a 6.7-kilobase pair DNA fragment derived from the wild-type K99 plasmid and the vector pBR322. The cloned K99 DNA expressed seven polypeptides with apparent masses of 18.2, 19.0, 21.0, 21.5, 26.5, 33.5, and 76.0 kilodaltons (kd). The 18.2-kd polypeptide was identified as the K99 fimbrial subunit by reaction with specific anti-K99 antibodies. The fimbrial subunit and the 19.0-, 26.5-, 33.5-, and 76.0-kd polypeptides appeared to be synthesized in a precursor form which was ca. 2 kd larger than the mature polypeptide. The location of the structural genes encoding the seven polypeptides on the physical map of pFK99 was established by analyzing a set of deletion derivatives of pFK99. The gene encoding the fimbrial subunit was located at the promoter proximal end of the K99 operon. Only mutants with a deletion in the gene encoding the 33.5- or the 19.0-kd polypeptide or both showed a weak expression of the K99 antigen and a comparably weak agglutination of horse or sheep erythrocytes. None of the deletion mutants was able to adhere to calf intestinal epithelial cells.