Genome-scale reconstruction of the Lrp regulatory network in Escherichia coli

Abstract

Broad-acting transcription factors (TFs) in bacteria form regulons. Here, we present a 4-step method to fully reconstruct the leucine-responsive protein (Lrp) regulon in Escherichia coli K-12 MG 1655 that regulates nitrogen metabolism. Step 1 is composed of obtaining high-resolution ChIP-chip data for Lrp, the RNA polymerase and expression profiles under multiple environmental conditions. We identified 138 unique and reproducible Lrp-binding regions and classified their binding state under different conditions. In the second step, the analysis of these data revealed 6 distinct regulatory modes for individual ORFs. In the third step, we used the functional assignment of the regulated ORFs to reconstruct 4 types of regulatory network motifs around the metabolites that are affected by the corresponding gene products. In the fourth step, we determined how leucine, as a signaling molecule, shifts the regulatory motifs for particular metabolites. The physiological structure that emerges shows the regulatory motifs for different amino acid fall into the traditional classification of amino acid families, thus elucidating the structure and physiological functions of the Lrp-regulon. The same procedure can be applied to other broad-acting TFs, opening the way to full bottom-up reconstruction of the transcriptional regulatory network in bacterial cells.

Fig. 1.

Overview of the method. (A) Comprehensive establishment of the Lrp-binding regions along with the changes in RNAP occupancies and mRNA transcript levels on a genome-scale to determine the regulatory mode for each of the identified Lrp-binding regions under leucine-perturbed growth conditions. (B) Determination of regulatory mode for individual ORFs governed by Lrp. (C) Reconstruction of regulatory network motif to identify logical motif structures composed of 2 feedback loops for transporting and metabolizing small molecules. (D) Understanding physiological behavior of regulatory network motifs.

Fig. 2.

Genome-wide distribution of Lrp-binding regions. (A) An overview of Lrp-binding profiles across the E. coli genome at exponential growth phase in the presence (i) or absence (ii) of leucine and at stationary growth phase (iii). Enrichment fold on the y axis was calculated from Cy5 (IP-DNA) and Cy3 (mock IP-DNA) signal intensity of each probe and plotted against each location on the 4.64 Mb E. coli genome. (B) Overlaps between Lrp-binding regions of exponential phase in the absence and presence of leucine, and stationary phase. (C) Comparison of the Lrp-binding regions obtained from this study (ChIP-chip) with the literature information. (D) Sequence logo representation of the learned Lrp-DNA binding profile.

Fig. 3.

Genome-wide rearrangement of RNAP occupancy. Upper and Lower show changes in RNAP- and Lrp-binding signals on the selected regions, (A) gcvTHP and serA, (B) oppABCDF, and (C) sdaA, respectively. Green (solid and dotted) lines and red (solid and dotted) lines in both Upper and Lower indicate the exponential growth condition in the absence and presence of the exogenous leucine, respectively. The dotted red and green lines in Upper indicate the RNAP binding on the promoter regions obtained from the rifampicin-treated cells. The triangle indicates the Lrp and RNAP binding location.

Fig. 4.

Regulatory modes of individual ORFs governed by Lrp in response to exogenous leucine. (A) Examples of independent mode [(I) gcvTHP and (II) ftsQAZ], concerted mode [(III) fimAICDFGH and (IV) livKHMGF], and reciprocal mode [(V) serA and (VI) oppABCDF]. Multiple lines indicate the Lrp-binding signals from the biological replicates. Green and red lines indicate the exponential growth condition in the absence and presence of the exogenous leucine, respectively. (B) Classification of the Lrp regulatory modes based on the gene expression profiling and location analysis of Lrp and RNAP.

Fig. 5.

Regulatory network motif reconstruction and the behavior of feedback loop motifs in response to the exogenous leucine. (A) Data integration between Lrp binding, RNAP occupancy, and gene expression profiles on the selected Lrp-regulated target genes (transporters and metabolic enzymes). (B) Schematic diagram for the regulatory network motif reconstruction in feedback loop. (C) Behavior of the feedback loop motifs.