BIOCHEMICAL-CHARACTERIZATION OF A HAEMOPHILUS-INFLUENZAE PERIPLASMIC IRON TRANSPORT OPERON

Bacterial iron transport is critical for growth of pathogens in the ho st environment, where iron is limited as a form of nonspecific immunit y. For Gram-negative bacteria such as Haemophilus influenzae, iron fir st must be transported across the outer membrane and into the periplas mic space, then from the periplasm to the cytosol. H. influenzae expre ss a periplasmic iron-binding protein encoded by the hitA gene. This g ene is organized as the first of a three-gene operon purported to enco de a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC ). In this study we describe the cloning, overexpression, and purifica tion of the H. influenzae hitA gene product. The function of this prot ein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in v itro and within the periplasmic space of a siderophore-deficient strai n of Escherichia coil. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing mediu m. These studies represent a detailed genetic, biochemical, and physio logic description of an active transport system that has evolved to ef ficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.