Seven strains of Actinobacillus suis (ATCC 15557, B49, C84, H89-1173, H91-0380, SO4 and VSB 3714) were investigated with respect to iron acquisition from animal transferrins (Tfs) and haemoglobins (Hbs). Growth assays with porcine, bovine and human Tfs and Hbs revealed that all seven strains could use porcine (but not human or bovine) Tf and all three Hbs as iron sources. In solid phase binding assays, membranes derived from all strains exhibited strong binding of porcine Tf and each of the Hbs. Competition binding assays indicated that all three Hbs were bound by the same receptor(s). Affinity procedures allowed the isolation and identification of iron repressible Tf-binding (~100 kDa and ~63 kDa) and Hb-binding (~105 kDa) polypeptides from all strains. Nucleotide sequence analyses revealed that A. suis strains SO4 and C84 possess genes that encode homologues of the Actinobacillus pleuropneumoniae Tf-binding proteins, TbpA and TbpB, and Hb-binding protein, HgbA. In both strains, tbpB was located immediately upstream of tbpA and was shown to be preceded by tonB, exbB and exbD homologues; hgbA was shown to be preceded by a hugZ homologue. Putative promoter and Fur box sequences were located upstream of tonB and hugZ and RT-PCR revealed that the genes in each of these clusters (tonB-exbB-exbD-tbpB-tbpA; hugZ-hgbA) are co-transcribed and iron-repressible. The molecular masses of the predicted mature TbpA, TbpB and HgbA proteins were calculated to be 104.3, 63.4 and 105.0 kDa, respectively, suggesting that the affinity-isolated, ~100 kDa and ~63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively, and that the ~105 kDa Hb-binding polypeptide represents HgbA. TbpB of A. suis was expressed in Escherichia coli and the recombinant TbpB (rTbpB) was identified by immunoblotting using swine sera raised against recombinant TbpB (A. pleuropneumoniae). It is envisaged that the acquisition of Tf- and Hb-bound iron by A. suis involves mechan
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85880 |
| Date | January 2005 |
| Creators | Bahrami, Fariborz |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Natural Resource Sciences.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002271880, proquestno: AAINR21617, Theses scanned by UMI/ProQuest. |
Page generated in 0.002 seconds