IDENTIFICATION OF A LOCUS INVOLVED IN THE UTILIZATION OF ICON BY HAEMOPHILUS-INFLUENZAE

Haemophilus influenzae has an absolute requirement for heme for aerobi c growth. This organism can satisfy this requirement by synthesizing h eme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme, In contra st, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme, A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DL42 the ability to grow on iron and PPIX. Nucleotide sequence analysis reveale d that this NTHI DNA insert contained three genes, designated hitA, hi tB, and hitC, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmi c binding protein-dependent iron transport system in this enteric orga nism. The NTHI HitA protein also was 69% identical to the ferric-bindi ng protein of Neisseria gonorrhoeae. Inactivation of the cloned NTHI h itC gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth defic iency of Hib DL42. Construction of an isogenic NTHI TN106 mutant tacki ng a functional hitC gene revealed that this mutation prevented this s train from growing on a medium containing iron and PPIX in place of he me. This NTHI hitC mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the produ cts encoded by the hitABC genes are essential for the utilization of i ron by NTHI.