Roles of the EnvZ/OmpR Two-Component System and Porins in Iron Acquisition in Escherichia coli

Abstract

Escherichia coli secretes high-affinity Fe³⁺ chelators to solubilize and transport chelated Fe³⁺ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe²⁺ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe²⁺ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon.IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ, which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Keywords: EnvZ/OmpR; ferric transport; ferrous transport; iron homeostasis; porins; two-component signal transduction systems.

FIG 1

Determination of fecA, fepA fhuA, and fhuF expression in different genetic backgrounds by real-time quantitative PCR (RT-qPCR). RNA was isolated from bacterial cultures grown to mid-log phase. Relative quantification of transcripts was performed using the 2^–ΔΔCT method, with ftsL and purC serving as the reference genes. The relative fold changes in gene expression and error bars representing standard deviations are shown. The bacterial strains used included RAM1292 (wild type), RAM1541 (envZ_R397L), RAM2697 (Δfur), RAM2698 (envZ_R397L Δfur), RAM2699 (ΔompC), RAM2700 (envZ_R397L ΔompC), RAM2701 (ΔfeoB), and RAM2702 (envZ_R397L ΔfeoB).

FIG 2

Determination of the relative gene expression of feoA and feoB by RT-qPCR. RNA was isolated from bacterial cultures grown to mid-log phase. Relative quantification of transcripts in various genetic backgrounds was performed using the 2^–ΔΔCT method, with ftsL and purC serving as reference genes. The relative fold changes in gene expression and error bars representing standard deviations are shown. The bacterial strains used included RAM1292 (wild type), RAM1541 (envZ_R397L), RAM2697 (Δfur), RAM2698 (envZ_R397L Δfur), RAM2699 (ΔompC), RAM2700 (envZ_R397L ΔompC), RAM2701 (ΔfeoB), and RAM2702 (envZ_R397L ΔfeoB).

FIG 3

Determination of the intracellular free iron concentration. The averages of five ferric-chelate EPR scans per strain are shown. All scans were normalized to the final culture OD₆₀₀ used in the measurements. The EPR parameters were as follows: microwave power, 10 mW; microwave frequency, 9.44 GHz; center field, 160 mT; sweep width, 80 mT; modulation amplitude, 1.25 mT; and modulation frequency, 100 kHz. The free intracellular iron concentrations, calculated as described in Materials and Methods, were as follows: wild type, 32 μM; Δfur, 120 μM; envZ_R397L, 135 μM; Δfur envZ_R397L, 105 μM; ΔfeoB, 20 μM; and ΔfeoB envZ_R397L, 29 μM. The bacterial strains used included RAM1292 (wild type), RAM1541 (envZ_R397L), RAM2697 (Δfur), RAM2698 (envZ_R397L Δfur), RAM2701 (ΔfeoB), and RAM2702 (envZ_R397L ΔfeoB).

FIG 4

Determination of fepA::lacZ and feo::lacZ activities in various genetic backgrounds. The β-galactosidase activities were measured from two independent overnight grown cultures. Error bars represent standard deviations. The bacterial strains used included RAM2920 (ompR⁺ envZ⁺ fepA::lacZ), RAM2921 (ompR⁺ envZ_R397L fepA::lacZ), RAM2922 (ompR_D55Y envZ_R397L fepA::lacZ), RAM2923 (ompR⁺ envZ⁺ feo::lacZ), RAM2924 (ompR⁺ envZ_R397L feo::lacZ), and RAM2925 (ompR_D55Y envZ_R397L feo::lacZ).

FIG 5

In vitro binding of purified OmpR_6His to the feoABC and ompC promoter regions. DNA binding was examined by EMSA using biotin-labeled DNA fragments of various lengths generated by PCR. (A) Diagram showing the regulatory region of the feoABC operon (not drawn to scale). Gray and black boxes represent possible OmpR binding sequences. Nucleotide numberings are relative to the feoA start codon. The relative positions and lengths of the two DNA fragments used in EMSA are shown. Diamond marks the biotin-labeled end of the DNA probe. (B) Diagram showing the regulatory region of the ompC gene (not drawn to scale). Three boxes represent the known OmpR binding sites; the DNA sequences of all three OmpR binding sites—C1, C2, and C3—are shown. Nucleotide numberings are relative the ompC start codon. The relative positions and lengths of the two DNA fragments used in the EMSA are shown. A diamond indicates the biotin-labeled end of the DNA probe. (C) Polyacrylamide gels showing EMSA results. Plus and minus signs denote the presence and absence, respectively, of OmpR in the reaction mixture prior to gel electrophoresis. Gels were electroblotted, and DNA bands were detected by treating membranes with stabilized streptavidin-HRP conjugate, followed by luminol/enhancer and stable peroxide. Arrows point to positions of unshifted DNA fragments.

FIG 6

Effects of ΔtonB and ΔaroB mutations on bacterial growth under iron-replete and iron-depleted conditions. Bacterial growth on LBA plus 40 μM FeCl₃ (A), LBA (B), and LBA plus 200 μM 2,2′-dipyridyl (C) was recorded after petri plates were incubated at 37°C for 24 h. Bacterial strains used: 1, RAM1292 (wild type); 2, RAM2553 (ΔaroB); 3, RAM2572 (ΔtonB); and 4, RAM2574 (ΔaroB ΔtonB).

FIG 7

Effects of ompR and porin gene mutations on the growth of ΔtonB or ΔtonB ΔaroB mutants. Bacterial growth was monitored on LBA plus 40 μM FeCl₃ (A and C) and LBA (B and D) after incubation of petri plates at 37°C for 24 h. Relevant genotypes of strains used in panels A and B: 1, RAM1292 (wild type); 2, RAM2572 (ΔtonB); 3, RAM2765 (ΔtonB malPQ::Tn10); 4, RAM2766 (ΔtonB malPQ::Tn10 ompR101); 5, RAM2767 (ΔtonB ΔompR::Km^r); 6, RAM2574 (ΔtonB ΔaroB::Km^r); 7, RAM2771 (ΔtonB ΔaroB::Km^r malPQ::Tn10); and 8, RAM2772 (ΔtonB ΔaroB::Km^r malPQ::Tn10 ompR101). Relevant genotypes of strains used in panels C and D: 1, RAM1292 (wild type); 2, RAM2572 (ΔtonB); 3, RAM2769 (ΔtonB ΔompC::Cm^r); 4, RAM2768 (ΔtonB ΔompF::Km^r); 5 and 6, no bacteria; 7, RAM2792 (ΔtonB ΔompC::Cm^r ΔompF::Km^r/pompC); and 8, RAM2790 (ΔtonB ΔompF::Km^r ΔompC::Cm^r/pompF). pompF and pompC are pTrc99A plasmid clones expressing ompF and ompC, respectively. The expression of these plasmid-coded genes did not require induction by an inducer.

FIG 8

Diagram showing regulation of the ferric (Fe³⁺) and ferrous (Fe²⁺) uptake systems in E. coli. Fur-Fe²⁺ is the master regulator of transcription of genes involved in iron metabolism. Under aerobic growth conditions, where Fe³⁺ is the major source of iron, E. coli secretes enterobactin (Ent) in the medium to chelate Fe³⁺. The Fe³⁺-chelate complex is transported back into the cell through the outer membrane receptor protein, FepA. The TonB-ExbB-ExbD complex of the inner membrane facilitates FepA channel opening. In the periplasm, FepB interacts with the Fe³⁺-chelate and delivers it to the FepDGC complex for transport into the cytoplasm. Under microaerobic or anaerobic growth conditions, Fe²⁺ is the main source of iron. It is brought into the cell via porins OmpC and OmpF and FeoB. The EnvZ/OmpR two-component system, classically known for regulating the expression of the ompC and ompF porin genes, also induces feo expression when hyperactivated due to a specific mutation in envZ (envZ_R397L). This positive effect of EnvZ_R397L/OmpR on feo expression can overcome the negative effect of Fur-Fe²⁺ on feo expression, thus tipping the balance in favor of ferrous over ferric transport. Porins and EnvZ/OmpR play a crucial role in iron acquisition in a TonB-deficient background that lacks functional ferric transport systems. Abbreviations: OM, outer membrane; PS, periplasm; IM, inner membrane; Cyt, cytoplasm; P, promoter.