CLONING AND SEQUENCING OF THE GENE ENCODING A 31-KILODALTON ANTIGEN OF HAEMOPHILUS-SOMNUS

Immunoblots using bovine antibody against Haemophilus somnus as the pr imary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. som nus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants exp ressed proteins which were recognized by bovine antiserum in Western b lots (immunoblots). Ten of the recombinants expressing a 31-kDa protei n caused the lysis of bovine erythrocytes. Restriction endonuclease ma pping indicated that the hemolytic recombinants shared an approximatel y 1.7-kb BglII fragment. Southern blot analysis using the BglII fragme nt as a probe revealed homology among the recombinants and the presenc e of an identically sized BglII fragment in the chromosome of all H. s omnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion o f this open reading frame resulted in the loss of hemolytic activity a nd protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequ ence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteria e, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonor rhoeae. An amino acid analysis of the recombinant 31-kDa protein agree d with the amino acid composition deduced from the DNA sequence.