Abstract
In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.
Highlights
To overcome the low availability of iron, most bacteria produce and utilize siderophores, small organic compounds that chelate ferric iron
An additional bacterial iron uptake permease, EfeU, was identified that belongs to the OfeT protein family (Transporter Classification Database entry 9.A.10.1) within the iron/lead transporter (ILT) superfamily (Transporter Classification Database entry 9.A.10) (5)
ChpA from the marine magnetotactic Vibrio strain MV-1 is required for magnetosome production (10); P19 from pathogenic Campylobacter jejuni is required for growth under iron limitation (11); Tp34 (TP0971) from Treponema pallidum is a lactoferrinbinding protein (12)
Summary
Vectors pGEM-T Easy pASK-IBA3 pET22b(ϩ) pTNS1 pUC18R6K-mini-Tn7T-Gm pUC18-mini-Tn7T-Gm-lacZ. Extraintestinal pathogenic E. coli (ExPEC), fetMPϪ W3110 ⌬lacZYA W3110 ⌬entC ⌬fecABCDE ⌬feoABC ⌬mntH ⌬zupT ECA458 glmS-Gm. ECA458 glmS-fetP-Gm W3110 ⌬lacZYA glmS-fetMp-lacZ recA1, endA1, gyrA96, thi-1, hsdR17, relA1, supE44, lac (F¢, proAB, lacIqZ_M15, Tn10) FϪ mcrA ⌬(mrr-hsdRMS-mcrBC) ⌽80dlacZ⌬M15 ⌬lacX74 recA1. Ref. 59 This study This study This study This study This study This study This study Stratagene (La Jolla, CA). FetM resembles EfeU fused to an N-terminal 39-kDa periplasmic protein with a potential membrane anchor or leader peptide. FetP (locus tag EcF11_1994, ZP_03035154), located directly downstream of fetM, encodes a putative periplasmic protein. We demonstrate that FetM constitutes a functional iron transport system that is enhanced by FetP. We have characterized the biochemical and structural properties of FetP, giving mechanistic insight into the potential role of copper binding in iron uptake
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