The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue

In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasteurella haemolytica grown under iron-d epleted conditions. This study reports identification of the gene (fbpA) de pleted conditions. This study reports identification of the gene (fbpA) enc oding the 35 kDa protein based on complementation of an entA Escherichia co li strain transformed with a plasmid derived from a P. haemolytica lambda Z AP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated O RF identified on the recombinant plasmid corresponded with the N-terminal a mino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding t he 35 kDa protein demonstrated homology with the cluster 1 group of extrace llular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert i dentified three other predominant ORFs, two of which appeared to be in an o peronic organization with fbpA. These latter components (fbpB and fbpC) sho wed homology to the transmembrane and ATPase components of ATP-binding cass ette (ABC)-type uptake systems, respectively. Based on amino acid/DNA seque ncing, citrate competition assay of iron affinity and visible wavelength sp ectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this p rotein is part of an operon comprising a member of the FbpABC family of iro n uptake systems. Primary sequence analysis revealed rather surprisingly th at PFbpA is more closely related to the intracellular Mn/Fe-binding protein IdiA found in cyanobacteria than to any of the homologous FbpA proteins cu rrently known in commensal or pathogenic members of the Pasteurellaceae or Neisseriaceae.