title: Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin.
abstract: The mechanism(s) used by Haemophilus influenzae to acquire the essential nutrient heme from its human host has not been elucidated. The heme carried by the high-affinity serum protein hemopexin is one potential source of this micronutrient in vivo. A colony-blot assay revealed that heme-human hemopexin-binding activity was shared among most capsular serotype b strains of H. influenzae but was uncommon among other strains. We have identified a recombinant clone binding heme-human hemopexin from a H. influenzae type b (Hib) genomic library expressed in Escherichia coli. Both the Hib strain and the heme-hemopexin-binding clone expressed a polypeptide of approximately 100 kDa that bound radiolabeled heme-hemopexin. Oligonucleotide linker insertion mutagenesis of the plasmid DNA from this recombinant clone was used to confirm that expression of the 100-kDa protein correlated with the heme-hemopexin-binding activity. Exchange of one of these mutant alleles into the Hib chromosome eliminated expression of both the 100-kDa protein and the heme-hemopexin-binding activity. Furthermore, this Hib mutant was unable to utilize heme-human hemopexin as a heme source.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: M S
authors.initials: MS
authors.lastname: Hanson
authors.forename: S E
authors.initials: SE
authors.lastname: Pelzel
authors.forename: J
authors.initials: J
authors.lastname: Latimer
authors.forename: U
authors.initials: U
authors.lastname: Muller-Eberhard
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D006418:Heme / Q000378:metabolism*; D006466:Hemopexin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006418:Heme; D006466:Hemopexin