title: TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA.
abstract: Siderophore nutrition tests withCaulobacter crescentusstrain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin.C. crescentusdid not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [59Fe]apoferrichrome and59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd], 1 nM; Michaelis-Menten constant [KM], 0.1 nM;Vmax, 19 pMol/109cells/min) were similar to those ofEscherichia coliFhuA. Transport properties for59Fe-citrate were similar to those ofE. coliFecA (KM, 5.3 nM;Vmax, 29 pMol/109cells/min). Bioinformatic analyses implicated Fur-regulated loci00028,00138,02277, and03023as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 toEscherichia coliFepA and BtuB. A Δ02277mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the02277structural gene ashutA(forheme/hemoglobinutilization).IMPORTANCEThe physiological roles of the 62 putative TBDT ofC. crescentusare mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems ofC. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition byC. crescentus, its heme/hemoglobin transporter (HutA).
authors.affiliation: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Heloise
authors.initials: H
authors.lastname: Balhesteros
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shipelskiy
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Noah J
authors.initials: NJ
authors.lastname: Long
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Aritri
authors.initials: A
authors.lastname: Majumdar
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Katz
authors.affiliation: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Naara M
authors.initials: NM
authors.lastname: Santos
authors.affiliation: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Laura
authors.initials: L
authors.lastname: Leaden
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Salete M
authors.initials: SM
authors.lastname: Newton
authors.affiliation: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Marilis V
authors.initials: MV
authors.lastname: Marques
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA peklebba@ksu.edu.
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Klebba
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D016935:Caulobacter crescentus / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007504:Iron Radioisotopes; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D017262:Siderophores
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D006454:Hemoglobins; D007504:Iron Radioisotopes; D008565:Membrane Proteins; D017262:Siderophores; C057616:tonB protein, Bacteria; D006418:Heme; D007501:Iron
keywords: TonB; alphaproteobacteria; heme transport; hemoglobin; iron acquisition; siderophores