During bacteriophage studies on Haemophilus influenzae, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal substance active against types a, c, d, e, and f H. influenzae, non-typable H. influenzae strains, other Haemophilus species, and certain members of the Enterobacteriaceae. The bactericidal activity was assayed by a plaque test utilizing an Rd strain as an indicator lawn and was also demonstrated in mixed broth cultures of a producer strain and an indicator strain. Immediate lysis of sensitive bacteria by the factor was not evident. The factor is sensitive to trypsin but resistant to deoxyribonuclease, treatment with 2-mercaptoethanol, lipase, α-amylase, and heating in a 100 °C water bath for 20 min. The activity is not dependent upon increased Ca2+ or Mg2+ concentration as is necessary for HP1C1 and S2 phage propagation. The bactericidal factor is not pelleted by high-speed centrifugation at 150 000 × g for 6 h. Treatment with ultraviolet light or mitomycin C does not result in observable phage, phage-like particles, or increased bactericidal activity. The bactericidal factor is not a typical small molecular weight "colicin-like" bacteriocin in that it is not inducible, has a wider range of activity, and does not kill by "single-hit" kinetics. On preliminary characterization, it is a thermostable protein toxic to certain bacterial strains.
The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species.
