B. L. Clark

Amino acid sequence of pilin from Bacteroides nodosus (strain 198), the causative organism of ovine footrot

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Analysis of the outer membrane proteins of Bacteroides nodosus, the causal organism of ovine footrot.

Examination by SDS-PAGE of lithium acetate extracts of several strains of depiliated Bacteroides nodosus revealed 6 major outer membrane proteins (including pilin). The 5 membrane proteins exhibited approximate molecular weights of 75000, 50000, 38000, 34500 and 26500 whereas pilin had a MW of 17500 for the majority of strains. All proteins were accessible to lactoperoxidase-catalysed iodination and proteins 1, 2 and 5 were shown to be glycoproteins. Several attempts to isolate individual OMC proteins in pure form by selective solubilization and gel filtration were unsuccessful, but electroelution of individual outer membrane complex proteins resolved by SDS-PAGE provided sufficient quantities of antigen for immunization of sheep and for immunochemical analysis.

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin‐producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.

Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with the corresponding protein from strain 198.

The amino acid sequence of pilin protein from Bacteroides nodosus strain 216 was determined. The protein had a calculated molecular weight of 15962 and contained the same number of amino acid residues (151) as the pilin from the previously sequenced strain 198. The sequence of the first 44 residues was common to both strains, including the unusual amino-terminal amino acid, N-methylphenylalanine. Of the remaining 107 residues, 37% of them differed between the two strains. Comparison of hydrophilicity profiles constructed from the sequence data indicated that a conserved region around residues 71-72 was probably the site of an antigenic determinant.