CLONING OF A DNA FRAGMENT ENCODING A HEME-REPRESSIBLE HEMOGLOBIN-BINDING OUTER-MEMBRANE PROTEIN FROM HAEMOPHILUS-INFLUENZAE

Haemophilus influenzae is able to use hemoglobin as a sole source of h eme, and heme-repressible hemoglobin binding to the cell surface has b een demonstrated. Using an affinity purification methodology, a hemogl obin-binding protein of approximately 120 kDa was isolated from H. inf luenzae type b strain HI689 grown in heme-restricted but not in heme-r eplete conditions. The isolated protein was subjected to N-terminal am ino acid sequencing, and the derived amino acid sequence was used to d esign corresponding oligonucleotides. The oligonucleotides were used t o probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridi zing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleoti de sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human he moglobin, whereas binding of hemoglobin was not detected in E. coli wi th the vector alone. In conclusion, we hypothesize that the DNA fragme nt encoding an approximately 120-kDa heme-repressible hemoglobin-bindi ng protein mediates one step in the acquisition of hemoglobin by H. in fluenzae in vivo.