IDENTIFICATION OF PASTEURELLA-MULTOCIDA TRYPTOPHAN SYNTHASE BETA-SUBUNIT BY ANTISERA AGAINST STRAIN P1059

Pasteurella multocida strain P1059 is a highly virulent bacterium whic h causes fowl cholera in turkeys and chickens. A genomic library of P. multocida P1059 DNA was constructed using pUC19, expressed in Escheri chia coli DH5 alpha, and screened with chicken antisera generated agai nst P. multocida P1059. Twelve out of the 4100 clones screened were im munoreactive. Plasmids isolated from these twelve clones were transfor med into E. coli CSR603 for maxicell analysis. Five proteins, with mol ecular masses of 34, 37, 43, 46 and 55 kDa, were expressed, Further wo rk focused on the 43 kDa protein because it was expressed at levels de tectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was de termined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The se cond 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 o bserved in the P. multocida enzyme, including the conserved lysine (Ly s-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphat e binding. The expression and identity of the P. multocida TrpB were c onfirmed by complementation studies using E. coil W3110 tnaA2 trpB9578 . The third ORF (ORF3) consisted of the first 77 nucleotides of the ge ne encoding the alpha-subunit of tryptophan synthase (trpA), and overl apped the 3'-end of trpB by 14 nucleotides. The deduced amino acid seq uence of the 77 nucleotides of the P. multocida TrpA had 68% identity (92% similarity) with the analogous region of TrpA from Klebsiella aer ogenes (K. pneumoniae).