When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (ϳ35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of ϳ5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33-35 kcal/mol.

The loops of the bacterial outer membrane iron transporter FepA move at different rates to adsorb and grasp the substrate ferric enterobactin before transporting it into the periplasm.

SummaryWe studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37°C. At 0°C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N-and Cdomains of FepA. However, colicin B penetration into the cell at 37°C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonBdependent structural changes in FepA, either alone or when it interacted with colicin B at 37°C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.

Kinases represent an important class of targets for pharmaceutical drug development. Microfluidic devices capable of running kinase assays with either an on-chip or an off-chip enzymatic reaction have been developed. For the on-chip assay, reagent addition, mixing, enzymatic reaction, and electrophoretic separation and detection of substrate and product all take place in the channels of the microfluidic chip. For the off-chip assay, the reaction takes place in a microtiter plate, whereas the electrophoretic separation and detection of substrate and product take place in the channels of the chip. To probe differences between the on-chip and off-chip assays, a panel of commercially available kinase inhibitors was assayed at 10 microM against cyclic AMP-dependent protein kinase A, glycogen synthase kinase 3beta, mitogen- and stress-activated protein kinase, and Akt1 using both the off-chip and on-chip assays. Good correlation was observed between inhibition measured by the two methods, with most of the differences in measured inhibition being attributed to compound solubility and enzyme concentration effects. Microfluidic devices represent an attractive platform for kinase assays due to high data quality and the possibility of on-chip assay integration, leading to reagent and labor savings.

The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N–C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [59Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N–C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.