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Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae

Mario Jacques^2,

James W. Coulton^1,

¹ Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, QC, Canada H3A 2B4
² Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada J2S 7C6
³ MDS Pharma Services, St Laurent, QC, Canada H4R 2N6

Correspondence
James W. Coulton
james.coulton{at}mcgill.ca

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for –35 and –10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Mario Jacques and James W. Coulton are affiliated to the Canadian Research Network on Bacterial Pathogens of Swine.

The GenBank accession number for the sequence of hgbA from Actinobacillus pleuropneumoniae reported in this paper is AF468020.

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