Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli

Abstract

The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements. Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability.

Figure 1. Flowchart of the method

The in vivo genome-wide Fur-binding maps, RNAP binding profiles (S, static map; D, dynamic maps) and Fur-dependent transcriptomic data were generated under both iron-replete and iron starvation conditions. Combined data sets were used to determine direct Fur regulon and the regulatory mode for individual ORFs governed by Fur. The Fur regulatory network was reconstructed by connecting iron transport and utilization regulatory motifs with negative-feedback loops. The regulatory modes (apo-Fur repression) and motifs (AA/AR pair) that are not identified in this study were presented in translucent format.

Figure 2. Genome-wide distribution of Fur-binding sites

(a) An overview of Fur-binding profiles across the E. coli genome at mid-exponential growth phase under both iron-replete (red) and iron starvation (blue) conditions. Black and white dots indicate previously known and newly found Fur-binding sites, respectively. S/N denotes signal to noise ratio. (+) and (−) indicate forward and reverse reads, respectively. (b) Overlaps between Fur-binding sites under iron-replete and iron starvation conditions. (c) Comparison of the Fur-binding sites obtained from this study (ChIP-exo) with the literature information.

Figure 3. Genome-wide identification of Fur regulon

Comparison of ChIP–exo results and gene expression profiles under (a) iron-replete and (b) iron starvation conditions to distinguish direct and indirect Fur regulon. (c) Functional classification of genes directly regulated by Fur. The asterisk indicates Hypergeometric P-value < 0.05.

Figure 4. Regulatory modes of individual ORFs governed by Fur in response to iron availability

(a) Examples of holo-Fur repression (HR) mode (fepA-entD and fes-ybdZ-entF- fepE), holo-Fur activation (HA) mode (ftnB and ftnA), and apo-Fur activation (AA) mode (ycgZ- ymgA-ariR-ymgC). S/N denotes signal to noise ratio. (+) and (−) in ChIP-exo data indicate forward and reverse reads, respectively. Boxes with dotted lines are zoom-in examples in Supplementary Fig. 2. (b) Sequence logo representations of the Fur-DNA binding profiles with consensus sequence highlighted with arrows. H-Reg-R, holo-Fur repression; H-Reg-A, holo-Fur activation; H-Reg-N, holo-Fur binding peaks in regulatory regions but no change in transcript level; H-NoReg-N, holo-Fur binding peaks in non-regulatory regions; B-Reg-N, binding peaks regardless of iron availability in regulatory regions but no change in transcript; B-NoReg-N, binding peaks regardless of iron availability in non-regulatory regions.

Figure 5. Iron acquisition/utilization pathways directly regulated by Fur and regulatory network motif

(a) The iron acquisition (enterobactin biosynthesis and iron/enterobatin transport), iron utilization (iron storage and iron/FeS cofactors), and FeS assembly pathways are represented. The genes regulated by HR and HA are depicted by red and blue characters, respectively. The genes regulated by RyhB-mediated mRNA degradation are depicted by green characters with black boxes. 2,3-DHBA, 2,3-dihydroxybenzoic acid; IM, inner membrane; OM, outer membrane. (b) Schematic diagram for the Fur regulatory motif reconstruction for iron metabolism. Shaded grey boxes indicate motif loops utilized by E. coli K-12 MG1655 for iron metabolism (negative/negative feedback loop motifs achieved by HR/HA modes of regulation).

Figure 6. Global coordination roles of the Fur regulatory network in E. coli

The Fur regulatory network is involved in many cellular functions required in addition to iron acquisition and utilization. Fur directly regulates genes associated with (a) iron metabolism, (b) DNA synthesis, (c) redirection of metabolism toward fermentative pathways, and (d) biofilm formation for searching nutrients in response to iron availability. These networks are linked through the coordination role that Fur plays. Bold characters indicate direct targets for Fur regulation and underlined characters indicate novel Fur regulon revealed in this study.