Molecular cloning and characterization of the genetic determinant encoding CS3 fimbriae of enterotoxigenic Escherichia coli.

The genetic determinant encoding the synthesis and surface expression of CS3 fimbriae of colonization factor antigen II-(CFA/II-) positive enterotoxigenic Escherichia coli was cloned on a 5.1 kb HindIII DNA fragment in pBR322 from the wild-type plasmid pCS001 to yield the CS3+ plasmid pCS100. Subcloning of EcoRI fragments of 1.8 kb and 2.5 kb into vector plasmid pACYC184 and the isolation of a series of pCS100::Tn5 insertion mutants revealed that more than one cistron was involved in the synthesis and expression of CS3 fimbriae. Polypeptides of 94, 26, 24, 17 and 15 kDa were detected in E. coli minicells harbouring pCS100. In Western immunoblotting the 17 kDa and 15 kDa polypeptides reacted with specific anti-CS3 fimbriae serum. The 15 kDa polypeptide comigrated with the structural subunit of CS3 fimbriae. Inhibition of protein processing in minicells by ethanol confirmed that the 17 kDa polypeptide was the precursor form of the 15 kDa structural subunit. A physical map of the cloned DNA was constructed showing the location and direction of transcription of the genes for the 17 and 94 kDa polypeptides. Using the 5.1 kb HindIII fragment of pCS100 as a genetic probe for the CS3 determinant, Southern hybridization analysis of plasmid and total cellular DNA was performed in wild-type enterotoxigenic E. coli strains.