Characterization of hgpA, a gene encoding a haemoglobin/haemoglobin-haptoglobin-binding protein of Haemophilus influenzae

Haemophilus influenzae binds haemoglobin and the haemoglobin-haptoglobin co mplex and utilizes either as a sole source of haem. Previously, a DNA fragm ent was cloned from H. influenzae that encodes an approximately 120 kDa pro tein (HgpA) expressing haemoglobin-binding activity in Escherichia coli. Pa rtial sequence analysis revealed significant homology of HgpA with other ba cterial haem- and iron-utilization proteins, and a length of CCAA repeating units immediately following the nucleotide sequence encoding the putative leader peptide. In the present study, the complete nucleotide sequence of t he cloned DNA fragment was determined and the sequence was analysed. In add ition to homology with other haem- and iron-utilization proteins, seven reg ions typical of TonB-dependent proteins were identified. The transcript of hgpA was determined to be monocistronic by RT-PCR. PCR performed with diffe rent colonies of a single H. influenzae strain at one CCAA-repeat-containin g locus indicated varying lengths of CCAA repeats, suggesting that haemoglo bin and haemoglobin-haptoglobin binding in H. influenzae is regulated by st rand slippage across CCAA repeats, as well as by haem repression. E. coli c ontaining cloned hgpA bound both haemoglobin and the haemoglobin-haptoglobi n complex. A deletion/insertion mutation of hgpA was constructed in H. infl uenzae strain H1689. Mutation of hgpA did not affect the ability of H. infl uenzae either to bind or to utilize haemoglobin or haemoglobin-haptoglobin following growth in haem-deplete media. Affinity purification of haemoglobi n-binding proteins from the mutant strain revealed loss of the 120 kDa prot ein and an increased amount of a 115 kDa protein, suggesting that at least one additional haemoglobin-binding protein exists.