title: The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants.
abstract: The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe(3+) transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe(3+) through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled (55)Fe(3+) transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Lothrop Street, Pittsburgh, PA 15261, USA.
authors.forename: Damon S
authors.initials: DS
authors.lastname: Anderson
authors.forename: Pratima
authors.initials: P
authors.lastname: Adhikari
authors.forename: Katherine D
authors.initials: KD
authors.lastname: Weaver
authors.forename: Alvin L
authors.initials: AL
authors.lastname: Crumbliss
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Mietzner
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D004252:DNA Mutational Analysis; D005708:Gallium / Q000494:pharmacology; D005998:Glycine / Q000235:genetics; D006193:Haemophilus influenzae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007930:Leucine / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017386:Sequence Homology, Amino Acid
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D005708:Gallium; D007501:Iron; D007930:Leucine; D005998:Glycine