1. Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.
- Author
-
Srikumar R, Mikael LG, Pawelek PD, Khamessan A, Gibbs BF, Jacques M, and Coulton JW
- Subjects
- Actinobacillus pleuropneumoniae metabolism, Amino Acid Sequence, Animals, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins metabolism, Base Sequence, Carrier Proteins chemistry, Carrier Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Gene Deletion, Molecular Sequence Data, Recombinant Proteins genetics, Recombinant Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Actinobacillus pleuropneumoniae genetics, Bacterial Outer Membrane Proteins genetics, Bacterial Proteins, Carrier Proteins genetics, Cell Membrane metabolism, Cloning, Molecular, Hemoglobins metabolism
- Abstract
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 approximately 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.
- Published
- 2004
- Full Text
- View/download PDF