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 leucineresponsive 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. ChIP-chip ͉ transcription factorT ranscriptional regulatory systems often regulate the formation rates and the concentration of small molecules by 2 feedback loops that regulate the transporters and metabolic enzymes. In many cases, these 2 feedback loops are connected by a common transcription factor (TF) that senses the concentration of the small molecule (1). Little is known at present about the transition between the regulatory modes in the feedback loop motifs for global TFs in bacteria. One such transcription factor is the leucineresponsive protein (Lrp), which is a global transcription regulator widely distributed throughout the bacteria including Escherichia coli (2-4). The Lrp regulon includes genes involved in amino acid biosynthesis and degradation, small molecule transport, pili synthesis, and other cellular functions including 1-carbon metabolism (2, 4-6). The regulatory action of Lrp on target genes is often modulated by the binding of the small effector molecule leucine and in effect endows Lrp with the ability to affect transcriptional regulation in all possible ways. That is, upon addition of leucine to the environment, the activity of Lrp can be enhanced, reversed, or unaffected (2, 4, 7). Little is known about in vivo Lrp-binding events at the genome scale in the presence or absence of leucine and the extent to which the different modes of regulation are used for different metabolites. Such information is needed to reconstruct the Lrp regulon and the understanding of nitrogen metabolism.In this study, we applied a systems approach by integrating genome-scale data from chromatin immunoprecipitation followed by microarray hybridization (ChIP-chip) for Lrp and...

Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp

“…Likewise, a shift to a lower antibiotic concentration for the ⌬hns and ⌬lrp backgrounds harboring pBAD-tosR-His 6 does not perturb TosR regulation itself; the lower antibiotic concentration did not perturb TosR-mediated regulation in the wild-type E. coli CFT073 background (data not shown). As for H-NS, Lrp is also a global regulator (19,22,(37)(38)(39)(59)(60)(61)(62)(63)(64)(65)(66)(67). Therefore, it remains unclear if additional gene products also supplement Lrp-and TosRmediated positive regulation of the tos operon.…”

“…The tos operon is localized to the PAI-aspV pathogenicity island in UPEC strain CFT073 (12). It is well accepted that genes on PAIs and other AT-rich sequences are often bound and regulated by nucleoid-structuring proteins, including H-NS and Lrp (41,43,44,67,79). A 400-bp region containing P tos is AT rich (74%).…”

Mechanisms and Evolution of Control Logic in Prokaryotic Transcriptional Regulation

“…Initial methodological tests reveal that especially a combination of traditional ChIP-onchip and ChIP sequencing yields a more comprehensive list of functional TFBSs throughout genomes (85). Also, combining high-resolution ChIP-on-chip or ChIP-Seq data with gene expression data appears to be promising for the network reconstruction of specific regulons (51). The integration of transcriptomics and metabolomics will finally also reveal more insights into the role of small-molecule concentrations (for example, as small TF binding ligands or in riboswitch regulation) in regulating gene transcription on a global scale (202), which is especially important when integrating experimental data for organisms grown in different media.…”

Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks

“…Before microarray hybridization, real-time quantitative PCR targeting previously known binding regions were carried out to verify enrichment of IP DNA fragments. qPCR and amplification of DNA was performed as previously described 8 . Microarray hybridization, wash and scan were performed in accordance with manufacturer's instruction (Roche Nimblegen).…”

“…These computational methods, however, do not account for condition-specific binding information, and thus cannot determine the functional state of the network. Therefore, experimental methods, such as chromatin immunoprecipitation coupled to microarray (ChIP-chip) or to sequencing (ChIP-seq), have recently been applied to determine condition-specific binding of s factors to DNA directly 7,8 .…”