Purification and Structural and Functional Characterization of FhuA, a Transporter of theEscherichia coliOuter Membrane

Abstract

The Escherichia coli outer membrane ferrichrome transporter FhuA was purified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside). The amount of dodecyl maltoside bound to the protein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodecyl maltoside complex (Rs = 4.2 nm) were determined using size exclusion chromatography. Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the protein was monomeric. The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for the complex. Sedimentation experiments gave values for the molecular mass of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes radius in complete agreement with those calculated from size exclusion chromatography. The circular dichroism spectrum indicated a 51% beta-sheet content. Functionality of the purified protein was assessed from fluorescence measurements using the DNA probe YO-PRO-1. Interaction of nM concentrations of FhuA with bacteriophage T5 resulted in the release of 90 +/- 8% of the phage DNA. The limiting step in DNA ejection was binding of the phage to its receptor. Release of DNA took place in a few seconds. Ferrichrome (0.8 microM) competed with the phage for binding to FhuA and prevented DNA ejection.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

“…Previous experiments [29] have shown that phage T5 ejects its DNA upon binding to purified FhuA, and DNA release can be measured using the fluorescent DNA intercalant YO-PRO- V f measurements, we calculated that the proportion of adsorbed phage decreased from 100 to 45 % when increasing the MccJ25 concentration from 0.1 to 3.2 µM (Figure 3). These values are in good agreement with the in vivo inhibition data (Figure 3).…”

“…MccJ25 and FhuA were purified as described previously [2,28] and quantified by amino acid composition analysis as in [21] , and peptide quantification was performed by amino acid composition analysis as described previously [29]. Peptide antibacterial activity was finally controlled according to the protocol described below.…”

“…After equilibration of the FhuA/MccJ25 mixture in the spectrofluorimeter (10 min, 37 • C), phage T5 (2.5 × 10 9 p.f.u./ml) was added and DNA release was monitored over 10 min by fluorescence spectroscopy using a SLM8000 spectrofluorimeter (λ excitation 490 nm; λ emission 509 nm). The rate of fluorescence increase (V f ), which is directly proportional to the number of phage bound to FhuA in the linear part of the ejection curve, was calculated between 50 and 100 s after the addition of phage T5 [29]. The percentage of adsorbed phage in the presence of microcin (50 nM-3.1 µM) was calculated by comparing the measured V f with a 100 % reference, determined in the absence of microcin.…”

DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes

“…The electron micrograph shown in Fig. 3C indicated that the toroids formed were rather homogenous in size, with an outer diameter of 300 nm, and that a comparatively constant number of phages (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) are involved in each structure.…”

“…Our strategy relies on the observation that the binding of bacteriophage T5 to its bacterial membrane receptor FhuA triggers the release of its genome, a double strand of 120,000 bp, linearly into the surrounding medium (15). Moreover, when FhuA is reconstituted in the membrane of a liposome, T5 phage bound to FhuA transfers its DNA directly into the liposome (16)(17).…”