GENETIC-ANALYSIS OF THE GENE-CLUSTER ENCODING NONFIMBRIAL ADHESIN-I FROM AN ESCHERICHIA-COLI UROPATHOGEN

The chromosomally encoded nonfimbrial adhesin I (NFA-I) from Escherich ia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination o f human erythrocytes. Subclones were constructed from an NFA-I-express ing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of appro ximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature i nto a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electr on microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the Nfa A precursor, which is processed to the mature protein, was found; it c onsisted of 156 amino acids with a calculated molecular weight of 16,0 00. Peptide sequencing of the NFA-I subunit protein confirmed that thi s open reading frame corresponds to the NfaA coding locus. Furthermore , the nucleotide sequence of the open reading frame termed NfaE, locat ed at the proximal part of the DNA stretch responsible for NFA-I expre ssion, was elaborated. NfaE consists of 247 amino acids, including a p resumptive 29-aminoacid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology wi th the 27-kDa CS3 protein, which is involved in the assembly of CS3 fi brillae, and might encode the 30.5-kDa protein, detected in minicells.