RESUMEN
The role of the penultimate and conserved tyrosine residue of the K99 major fibrillar subunit (FanC) in fibrillae biosynthesis and functioning was investigated. By using oligonucleotide-directed in vitro mutagenesis the TAT codon of tyrosine-158 of fanC was changed into a TAG stop codon. The mutant fanC gene encoded a truncated major subunit lacking the two carboxyl-terminal amino acid residues. Furthermore, the tyrosine residue (position 158) was replaced by a serine residue or by a glutamic acid residue. The effect of these mutations on the expression and binding capacity of K99 fibrillae was investigated by using an ELISA, an haemagglutination assay, Escherichia coli minicells and suppressor strains. All mutations completely blocked K99 fibrillae biosynthesis and haemagglutination activity. The mature form of the truncated mutant FanC polypeptide could not be detected in minicells, but its precursor was expressed at a normal level. The results showed that the penultimate tyrosine residue is essential for the expression of mature fibrillar subunits and suggested a function in the interaction with the periplasmic transport protein FanE.
Asunto(s)
Antígenos de Superficie/metabolismo , Toxinas Bacterianas , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Antígenos de Superficie/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Escherichia coli/genética , Escherichia coli/inmunología , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/metabolismo , Datos de Secuencia Molecular , Mutación , Tirosina/genética , Tirosina/metabolismoRESUMEN
Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.