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

Abstract

In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasfeurella haemolytica grown under irondepleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATPbinding cassette (ABC)-type uptake systems, respectively. Based on amino acicUDNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, 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 protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Febinding protein ldiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Past eurellaceae or Neisseriaceae .~~

Crystal Structure of Pasteurella haemolytica Ferric Ion-binding Protein A Reveals a Novel Class of Bacterial Iron-binding Proteins

“…Although literature describing biophysical characterizations of PhFbpA is sparse, the limited information available supports our claims. The iron-loaded protein possesses a peak visible absorbance ( max ) at 419 nm, which is dramatically blue-shifted when compared with the same parameter from HiFbpA and Tf ( max of ϳ480 nM) (15). Because the relative effects of anions on the absorbance maximum are more subtle (Ϫ10 to Ϫ15 nM) (35), the large change in the absorbance maximum in PhFbpA can probably be ascribed to the fact that PhFbpA uses three tyrosine residues to coordinate iron versus the two utilized in all of the other structurally characterized FbpAs as well as in Tf and lactoferrin.…”

“…Despite its homology to FbpA molecules of other organisms, PhFbpA has a peak-visible absorbance at 419 nm, which is significantly blue-shifted relative to the peak absorbance for other FbpAs (13,17). PhFbpA shares approximately the same iron affinity as transferrin and other FbpA molecules based on citrate competition assays (15). Thus, current evidence indicates that PhFbpA binds and transports Fe 3ϩ ion, utilizing a unique iron-coordinating environment when compared with transferrin or any other biochemically characterized FbpA molecules.…”

“…The mic space (13)(14)(15) as the periplasmic component of the iron ABC transport system (16). The ABC family of transporters includes a broad and diverse group of import systems found in prokaryotes.…”

“…Identification of the gene encoding PhFbpA was based on complementation of an entA Escherichia coli strain transformed with a P. haemolytica ZAP II library-derived plasmid (15). Nucleotide sequence analysis of the fbpA gene confirmed homology with the cluster I group of the periplasmic binding protein family (15,16).…”

“…Identification of the gene encoding PhFbpA was based on complementation of an entA Escherichia coli strain transformed with a P. haemolytica ZAP II library-derived plasmid (15). Nucleotide sequence analysis of the fbpA gene confirmed homology with the cluster I group of the periplasmic binding protein family (15,16). Despite its homology to FbpA molecules of other organisms, PhFbpA has a peak-visible absorbance at 419 nm, which is significantly blue-shifted relative to the peak absorbance for other FbpAs (13,17).…”