HGPB, A GENE ENCODING A 2ND HAEMOPHILUS-INFLUENZAE HEMOGLOBIN-BINDINGAND HEMOGLOBIN-HAPTOGLOBIN-BINDING PROTEIN

Haemophilus influenzae requires heme for growth and can utilize both h emoglobin and hemoglobin-haptoglobin as heme sources. We previously id entified a hemoglobin- and hemoglobin-haptoglobin-binding protein, Hgp A, in H. influenzae HI689, Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in bin ding of these heme sources by H. influenzae. A 3.2-kbp PCR product der ived from HI689 was cloned. The nucleotide sequence indicated a separa te, distinct gene with high homology to hgpA, which would encode a 115 -kDa protein. Primers were designed for directional cloning of the str uctural gene in the correct reading frame, Sonicates of induced Escher ichia coli harboring the cloned open reading frame bound both hemoglob in and hemoglobin-haptoglobin. An insertion/deletion mutant of H. infl uenzae at the newly identified locus, designated hgpB, was constructed . The 115-kDa protein was not detected in the mutant after affinity pu rification using biotinylated hemoglobin. An hgpA hgpB double-mutant s train exhibited a reduced ability to utilize hemoglobin-haptoglobin, a lthough it was unaltered in the ability to utilize hemoglobin, Affinit y isolation of hemoglobin-binding proteins from the double mutant resu lted in isolation of an approximately 120-kDa protein. Internal peptid e sequencing revealed this protein to be a third distinct protein, hig hly homologous to HgpA and HgpB. In summary a second hemoglobin- and h emoglobin-haptoglobin-binding protein of H. influenzae has been identi fied and characterized, and the presence of an additional protein of s imilar function has been revealed.