A FUNCTIONAL TONB GENE IS REQUIRED FOR BOTH UTILIZATION OF HEME AND VIRULENCE EXPRESSION BY HAEMOPHILUS-INFLUENZAE TYPE-B

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components i nvolved in the uptake of heme. Chemical mutagenesis was employed to pr oduce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This m utant was transformed with a plasmid shuttle vector-based genomic libr ary constructed from the same wild-type nontypeable H. influenzae stra in, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recom binant plasmid revealed the presence of several open reading frames, o ne of which encoded a 28-kDa protein with significant similarity to th e TonB protein of Escherichia coli. This H. influenzae gene product wa s able to complement a tonB mutation in E. call, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitam in B-12. When this H. influenzae gene was inactivated by insertional m utagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost thei r abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization abi lity and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal mo del.