Identification of Pasteurella multocida tryptophan synthase beta-subunit by antisera against strain P1059.

Pasteurella multocida strain P1059 is a highly virulent bacterium which causes fowl cholera in turkeys and chickens. A genomic library of P. multocida P1059 DNA was constructed using pUC19, expressed in Escherichia coli DH5 alpha, and screened with chicken antisera generated against P. multocida P1059. Twelve out of the 4100 clones screened were immunoreactive. Plasmids isolated from these twelve clones were transformed into E. coli CSR603 for maxicell analysis. Five proteins, with molecular masses of 34, 37, 43, 46 and 55 kDa, were expressed. Further work focused on the 43 kDa protein because it was expressed at levels detectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was determined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The second ORF (ORF2) encoded a protein of 403 amino acids (43,662 Da). The deduced amino acid sequence showed 77% identity (84% similarity) with the tryptophan synthase beta subunit (TrpB) of Salmonella typhimurium and Vibrio parahaemolyticus. The eight conserved regions of TrpB are observed in the P. multocida enzyme, including the conserved lysine (Lys-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphate binding. The expression and identity of the P. multocida TrpB were confirmed by complementation studies using E. coli W3110 tnaA2 trpB9578. The third ORF (ORF3) consisted of the first 77 nucleotides of the gene encoding the alpha-subunit of tryptophan synthase (trpA), and overlapped the 3'-end of trpB by 14 nucleotides. The deduced amino acid sequence of the 77 nucleotides of the P. multocida TrpA had 68% identity (92% similarity) with the analogous region of TrpA from Klebsiella aerogenes (K. pneumoniae).