title: Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae.
abstract: The bacterium Actinobacillus pleuropneumoniae, a swine pathogen, utilizes ferrichrome as an iron source. This study details the molecular cloning and sequencing of the genes involved in the uptake of this hydroxamate siderophore. Four ferric hydroxamate uptake (fhu) genes, fhuC, fhuD, fhuB and fhuA, were identified in a single operon, and these were found to encode proteins homologous to proteins of the fhu systems of several bacteria, including Escherichia coli. The fhuA gene encodes the 77 kDa outer-membrane protein (OMP) FhuA, the receptor for ferrichrome. FhuD is the 35.6 kDa periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane. FhuC (28.5 kDa) and FhuB (69.4 kDa) are cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes the ferric hydroxamate. Reference strains of A. pleuropneumoniae that represented serotypes 1 to 12 of this organism all tested positive for the four fhu genes. When A. pleuropneumoniae FhuA was affinity-tagged with hexahistidine at its amino terminus and expressed in an E. coli host, the recombinant protein reacted with an mAb against E. coli FhuA, as well as with a polyclonal pig serum raised against an A. pleuropneumoniae infection. Hence, the authors conclude that fhuA is expressed in vivo by A. pleuropneumoniae. Three-dimensional modelling of the OMP FhuA was achieved by threading it to the X-ray crystallographic structure of the homologous protein in E. coli. FhuA from A. pleuropneumoniae was found to have the same overall fold as its E. coli homologue, i.e. it possesses an N-terminal cork domain followed by a C-terminal beta-barrel domain and displays 11 extracellular loops and 10 periplasmic turns.
authors.affiliation: Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, CanadaJ2S 7C61.
authors.forename: Leonie G
authors.initials: LG
authors.lastname: Mikael
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, CanadaH3A 2B42.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Pawelek
authors.affiliation: Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, CanadaJ2S 7C61.
authors.forename: Josée
authors.initials: J
authors.lastname: Labrie
authors.affiliation: Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, CanadaG9A 5H73.
authors.forename: Marc
authors.initials: M
authors.lastname: Sirois
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, CanadaH3A 2B42.
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
authors.affiliation: Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, CanadaJ2S 7C61.
authors.forename: Mario
authors.initials: M
authors.lastname: Jacques
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D003001:Cloning, Molecular; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005796:Genes; D006877:Hydroxamic Acids / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; C050900:FhuA protein, E coli; D006877:Hydroxamic Acids; D011991:Receptors, Virus; C074579:iron (III) hydroxamate; D007501:Iron