A GENE-CLUSTER INVOLVED IN THE UTILIZATION OF BOTH FREE HEME AND HEME-HEMOPEXIN BY HAEMOPHILUS-INFLUENZAE TYPE-B

The utilization of heme bound to the serum glycoprotein hemopexin by H aemophilus influenzae type b (Hib) strain DL42 requires the presence o f the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide se quence analysis of a 5-kb region immediately upstream from the hxuA ge ne revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similari ty to TonB-dependent outer membrane proteins of other organisms, where as the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat car tridges inserted individually into their hxuA, hxuB, and hxuC genes wa s constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, where as both the hxuA and hxuB mutants could utilize free heme. When the wi ld type hxuC gene was present in trans, the hxuC mutant regained its a bility to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a fu nctional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. i nfluenzae hxuA gene also indicated that the HxuB protein likely functi ons in the release of soluble HxuA from the Hib cell. These studies in dicate that at least two and possibly three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.