The penultimate tyrosine residue of the K99 fibrillar subunit is essential for stability of the protein and its interaction with the periplasmic carrier protein.
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.
Simons
B L
BL
Department of Molecular Microbiology, Vrije Universiteit, Amsterdam, The Netherlands.
Rathman
P
P
Malij
C R
CR
Oudega
B
B
de Graaf
F K
FK
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001427:Bacterial Toxins; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; C031429:K99 antigen; D014443:Tyrosine