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Microbiology 149 (2003), 2273-2281; DOI  10.1099/mic.0.26370-0
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Microbiology 149 (2003), 2273-2281; DOI  10.1099/mic.0.26370-0
© 2003 Society for General Microbiology

fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein

M. Elena Garrido1, Montserrat Bosch1, Ricardo Medina1,{dagger}, Anna Bigas1, Montserrat Llagostera1,2, Ana M. Pérez de Rozas2, Ignacio Badiola2 and Jordi Barbé1,2

1 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 – Barcelona, Spain
2 Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona, Institut de Recerca i Tecnologia Agroalimentària (UAB-IRTA), Bellaterra, 08193 – Barcelona, Spain

Correspondence
Jordi Barbé
jordi.barbe@uab.es Ignacio Badiola
ignacio.badiola{at}irta.es

Treatment of bacterial cultures with chelating agents such as 2,2'-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the {gamma}-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the {gamma}-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.


Abbreviations: DPD, 2,2'-dipyridyl

{dagger}Present address: Centro de Bioactivos Químicos, Universidad Central de las Villas, Carretera Camajuani, km 5, Villa Clara, Cuba.




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