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Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae
- Source :
- Microbiology (Reading, England). 150(Pt 6)
- Publication Year :
- 2004
-
Abstract
- From the porcine pathogenActinobacillus pleuropneumoniaecultivated 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 tohgbAofPasteurella multocida. Upon screening two genomic libraries ofA. pleuropneumoniaeserotype 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 ofhgbAfromA. pleuropneumoniaeserotype 1 showed consensus for −35 and −10 sequences and a putative Fur-binding site. RT-PCR confirmed thathgbAofA. pleuropneumoniaeis upregulated in response to diminished levels of iron in the culture medium. While an internally deletedhgbAmutant 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 ofA. pleuropneumoniaein the presence of Hb as sole iron source.
- Subjects :
- Signal peptide
Mutant
Molecular Sequence Data
Molecular cloning
Microbiology
Hemoglobins
Bacterial Proteins
Escherichia coli
Animals
Genomic library
Amino Acid Sequence
Cloning, Molecular
Pasteurella multocida
Actinobacillus pleuropneumoniae
biology
Base Sequence
Reverse Transcriptase Polymerase Chain Reaction
Binding protein
Cell Membrane
Sequence Analysis, DNA
biology.organism_classification
Molecular biology
Recombinant Proteins
Biochemistry
Bacterial outer membrane
Carrier Proteins
Gene Deletion
Bacterial Outer Membrane Proteins
Subjects
Details
- ISSN :
- 13500872
- Volume :
- 150
- Issue :
- Pt 6
- Database :
- OpenAIRE
- Journal :
- Microbiology (Reading, England)
- Accession number :
- edsair.doi.dedup.....5891f38152f21c722850e3fe08db527b