We have determined that the DNA sequence downstream of the well-characterized gonococcal fbp gene contains two open reading frames: one designated fbpB, which encodes a protein proposed to function as a cytoplasmic permease, and one designated fbpC, which encodes a protein proposed to function as a nucleotidebinding protein. The fpbABC operon composes an iron transport system that is homologous to the sfu and hit operons previously reported for Serratia marcescens and Haemophilus influenzae, respectively, and displays elements characteristic of ATP binding cassette transporters. The fpbABC operon differs from these loci in that it is lethal when overexpressed in Escherichia coli.

Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic ironbinding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode 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 purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. 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 medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.High affinity acquisition of iron from the host environment is a necessary determinant of virulence for pathogenic bacteria (1-9). This acquisition is vital for survival in the human host, where levels of extracellular iron are tightly controlled by the Transferrins (transferrin and lactoferrin), a family of ironbinding proteins that function in the extracellular chelation and transport of host iron (9). By binding iron with high affinity, Transferrins ensure that all extracellular iron is both efficiently sequestered from pathogenic invaders and mobilized for transport to host tissues. Microorganisms growing in the human host must therefore possess mechanisms for obtaining Transferrin-sequestered iron. For a number of pathogenic members of the Pasteurellaceae (H. influenzae) and Neisseriaceae (Neisseria meningitidis and Neisseria gonorrhoeae), iron acquisition is initiated by cell-surface receptors specific for the Transferrins (10 -16). Iron is removed from these proteins and transported across the outer membrane, presumably by an energy-dependent TonB-mediated process (17-19) involving gated-pore properties of the outer membrane receptor (18,20). The result is deposition of free iron within the periplasm, where it is separated from the cytosol, its eventual destination, by the cytoplasmic membrane (21).Transport of free iron from the periplasmic space into the cytoplasm is proposed to occur by a classic active transport process involving a periplasmic binding protein, a specific cytoplasmic permease, and an energy-supplying nucleotide-binding protein (22). Much of what is known about the biochemistry of a...

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