Iron is an indispensable metabolic cofactor in both pro-and eukaryotes, which engenders a natural competition for the metal between bacterial pathogens and their human or animal hosts. Bacteria secrete siderophores that extract Fe 3+ from tissues, fluids, cells, and proteins; the ligand gated porins of the Gram-negative bacterial outer membrane actively acquire the resulting ferric siderophores, as well as other iron-containing molecules like heme. Conversely, eukaryotic hosts combat bacterial iron scavenging by sequestering Fe 3+ in binding proteins and ferritin. The variety of iron uptake systems in Gram-negative bacterial pathogens illustrates a range of chemical and biochemical mechanisms that facilitate microbial pathogenesis. This document attempts to summarize and understand these processes, to guide discovery of immunological or chemical interventions that may thwart infectious disease.

Iron uptake by Escherichia coli under aerobic conditions of iron deficiency is mediated by a highly stable ferric enterobactin [Fe(ent)3-] siderophore complex. Mossbauer spectroscopy has been used to monitor the fate of the iron as 57Fe(ent) was taken up by the cells. Osmotic shock experiments were used to distinguish between the iron present in the periplasmic space and that in the cytoplasm of the cell. Iron delivery by a synthetic analog of enterobactin, 1,3,5-N,N', N"-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was also studied. Although Fe-MECAM was transported at the same rate as was Fe(ent) across the outer membrane and was apparently accumulated in the periplasmic space, the subsequent behaviors of Fe(ent) and Fe-MECAM were very different. After more than 30 min, a major fraction of the iron originally absorbed as ferric enterobactin appeared as Fe(II), apparently in the cytoplasm of the cell. However, little iron was delivered to the cytoplasm by the MECAM complex. The differences in specificity of these two stages of iron uptake by E. coli are discussed.As described in our preceding paper (6), studies with synthetic analogs of enterobactin have established important correlations between the coordination chemistry of the ferric complex and recognition at the outer membrane of Escherichia coli. However, the use of radioactive labels or inhibition studies with synthetic analogs cannot monitor the oxidation state and coordination environment of the iron after the initial transport step. That is one purpose of the present study.A second purpose is to probe the mechanism of iron release by ferric enterobactin. Enterobactin possesses a cyclic backbone consisting of three ester-linked serine residues. As described in the introduction of the previous paper (6), whether iron release takes place primarily by reduction of Fe3+ to Fe2+ or rather by hydrolysis of enterobactin and subsequent dissociation of the ferric complex remains unresolved (3, 23). We have recently measured the reduction potential of the ferric enterobactin Fe(ent)3-in the pH range 6 to 11 (19). The potential at pH 6 is -0.56 V. Thus, reduction in an acidic environment of the cell remains a possible mechanism for iron removal from E. coli.Mossbauer data (25) have shown that (in vitro) the iron in ferric enterobactin remains as Fe(III) even after precipitation of the neutral [Fe(H3ent)]0 triprotonated complex. Mossbauer spectroscopy also has been employed to identify and study the diverse environments of iron in whole cells of procaryotes and eucaryotes (1,2,7,18,21,28,30). Here we apply this technique to obtain information concerning the mechanism of ferric enterobactin metabolism in the gramnegative bacterium E. coli.Intact E. coli cells contain two major cellular compartments: the cytoplasm and the periplasm (the volume contained between the outer and the inner membranes). The volume of the periplasm has been recently measured (31) and found to occupy 20 to 40% of the total cell volume, depending on the composition of the growth ...

Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. We created fluorescent sensors that monitor high-affinity binding reactions and used them to study iron acquisition by ESKAPE bacteria, which are frequently responsible for antibiotic-resistant infections. By introducing site-directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells or purified proteins that bind but do not transport target compounds. These constructs sensitively detected ligand concentrations in solution, enabling accurate, real-time spectroscopic analysis of membrane transport by other cells. We assessed the efficacy of these "fluorescent decoy" (FD) sensors by characterizing active iron transport in the ESKAPE bacteria. The FD sensors monitored uptake of both ferric siderophores and hemin by the pathogens. An FD sensor for a particular ligand was universally effective in observing the uptake of that compound by all organisms we tested. We adapted the FD sensors to microtiter format, where they allow high-throughput screens for chemicals that block iron uptake, without genetic manipulations of the virulent target organisms. Hence, screening assays with FD sensors facilitate studies of mechanistic biochemistry, as well as discovery of chemicals that inhibit prokaryotic membrane transport. With appropriate design, FD sensors are potentially applicable to any pro-or eukaryotic high-affinity ligand transport process.

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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.