title: Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.
abstract: Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.
authors.affiliation: Departments of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Morton
authors.forename: P W
authors.initials: PW
authors.lastname: Whitby
authors.forename: H
authors.initials: H
authors.lastname: Jin
authors.forename: Z
authors.initials: Z
authors.lastname: Ren
authors.forename: T L
authors.initials: TL
authors.lastname: Stull
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D019741:Haemophilus influenzae type b / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017422:Sequence Analysis, DNA
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; C119573:HgpC protein, Haemophilus influenzae; C017206:hemoglobin-binding protein, bacteria