Journal of Molecular Biology
Volume 356, Issue 5, 10 March 2006, Pages 1073-1081
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The Incoherent Feed-forward Loop Accelerates the Response-time of the gal System of Escherichia coli

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Complex gene regulation networks are made of simple recurring gene circuits called network motifs. One of the most common network motifs is the incoherent type-1 feed-forward loop (I1-FFL), in which a transcription activator activates a gene directly, and also activates a repressor of the gene. Mathematical modeling suggested that the I1-FFL can show two dynamical features: a transient pulse of gene expression, and acceleration of the dynamics of the target gene. It is important to experimentally study the dynamics of this motif in living cells, to test whether it carries out these functions even when embedded within additional interactions in the cell. Here, we address this using a system with incoherent feed-forward loop connectivity, the galactose (gal) system of Escherichia coli. We measured the dynamics of this system in response to inducing signals at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the galactose system displays accelerated turn-on dynamics. The acceleration is abolished in strains and conditions that disrupt the I1-FFL. The I1-FFL motif in the gal system works as theoretically predicted despite being embedded in several additional feedback loops. Response acceleration may be performed by the incoherent feed-forward loop modules that are found in diverse systems from bacteria to humans.

Introduction

Cells respond to external signals by means of transcription regulation networks.1, 2, 3, 4, 5, 6, 7, 8, 9 These networks are made of recurring regulatory circuits called network motifs.10, 11 Network motifs were first defined in the transcription network of Escherichia coli.10 The same set of motifs were then found to appear in the transcription networks of diverse organisms, including yeast,11, 12, 13 Bacillus subtilis,14, 15 Drosophila14 and humans.16, 17 Each network motif has been suggested to perform a key information processing function.10, 18, 19 It is important to experimentally study the function of each network motif in living cells, because this can shed light on the dynamical behavior of many systems across organisms that have the motif.

One of the most significant network motifs is the feed-forward loop (FFL).10, 11, 18, 20, 21, 22, 23, 24, 53, 54 The FFL is made of two cascaded transcription factors that jointly regulate a gene. In the FFL, transcription factor X regulates transcription factor Y, and both jointly regulate gene Z. Since each of the three interactions of the FFL can be either activation or repression, there are eight types of FFLs (Figure 1), corresponding to the different combinations of positive and negative regulation for each interaction. The frequency of each FFL type was counted in transcription regulation databases for both E. coli and Saccharomyces cerevisiae.18 Two FFL types are more common than others. These are termed the coherent type-1 FFL (C1-FFL), which contains three positive interactions (that is, X and Y are both transcriptional activators), and the incoherent type-1 FFL (I1-FFL) in which X activates Y and Z while Y represses Z. Coherent and incoherent type 1 FFLs can appear in interesting combinations, for example cascaded I1-FFLs and C1-FFls govern the gene regulation system for sporulation in B. subtilis.15 Often, the same X and Y regulate several genes, Z1,…Zn, forming a multi-output FFL.25, 26

Theoretical analysis suggested that the C1-FFL motif robustly performs a dynamical function termed sign-sensitive delay.18 This delay depends on the input function at the Z promoter. For example, when X and Y are both needed to activate Z, forming a C1-FFL with an AND input-function at the Z promoter, the C1-FFL shows a delay of Z expression following induction of X activity, but no delay following deactivation of X. Thus the C1-FFL can filter out brief fluctuations that activate X. This function was experimentally demonstrated in living cells in the arabinose (ara) utilization system of E. coli.20 In another example, when either X or Y are sufficient to activate Z, forming a C1-FFL with an OR or SUM input-function at the Z promoter, a delay occurs after deactivation of X but not after activation. This function, which allows continuous expression even if the input signal is briefly lost, was experimentally demonstrated in living cells using the flagella class 2 system of E. coli.24

Here, we experimentally study the second common FFL type, the type-1 incoherent FFL (I1-FFL) (Figure 1). In this motif, X positively regulates Y and Z, whereas Y represses Z expression. Thus, the two regulators X and Y act in opposite signs to control Z, hence the name “incoherent FFL”. I1-FFLs regulate over 100 different genes in E. coli21 and yeast,18 comprising about one-third of the total number of FFLs in these organisms (Figure 1). It is much more common than any of the other three types of incoherent FFL (types 2, 3 and 4 incoherent FFL). Recently, an I1-FFL involving a micro-RNA has been identified in mammalian cells.27

Theoretical analysis suggested that the I1-FFL can perform response acceleration:18 expression of the output gene Z has a shorter response time, defined as the time to reach halfway to the steady-state level, than a corresponding “simple regulation” system that reaches the same steady state. To see this, note that when X is induced, Z begins to be expressed. In parallel, the level of the repressor Y also increases. When Y activity exceeds the repression threshold for the Z promoter, Z expression is repressed, and drops to a low steady-state level. The result is an overshoot dynamics, which shortens the response-time. The response-time of Z in the I1-FFL is smaller than the response time of a corresponding simply regulated system in which X regulates Z without an I1-FFL. In the simply regulated system, the dynamics28 are exponential convergence to a steady state xst, such that x/xst=1−e−αt. The response time for stable proteins (not actively degraded in the cell) has been shown to be about one cell-generation time.28, 29 In contrast, the I1-FFL shows accelerated responses, by using a strong promoter to achieve rapid initial induction and a repressor to reduce production at a delay and reach the desired steady state. When the biochemical parameters are such that Y fully represses Z, the resulting dynamics resembles a pulse of Z expression.18

The I1-FFL was recently constructed as a synthetic gene circuit in E. coli by Weiss and colleagues.30 Well-studied transcription factors were connected in an I1-FFL pattern with a strong repression of Z by Y, whose readout was green-fluorescent protein. These experiments demonstrated that the I1-FFL can generate a pulse of expression following induction of X.

Both synthetic gene circuit experiments and the theoretical models deal with the I1-FFL as a three-gene circuit in isolation. In the real cell, this motif is embedded inside a network of additional interactions. It is therefore important to experimentally study its function in a natural system in living cells.

Here, we examine the dynamics of the I1-FFL in living cells using the well-studied crp-galS-galE system of E. coli,31, 32, 33, 34, 35, 36 which has an I1-FFL structure (Figure 2(a)). The gal system allows E. coli to grow on the sugar galactose. Expression of the gal genes is inhibited in the presence of glucose, a superior energy source. In the gal system, the galactose utilization operon galETK, called galE throughout this study, is transcriptionally regulated by CRP, an activator induced by glucose starvation. The galE promoter is also repressed by GalS. GalS unbinds from the galE promoter in the presence of the inducer β-d-galactose or its non-metabolizable analog d-fucose (Figure 2(a)), relieving the repression. The gene that encodes the repressor GalS is itself positively regulated by CRP, so that an I1-FFL is formed. Furthermore, GalS negatively regulates its own expression, forming a negative auto-regulation loop. This loop can act to speed28, 37 and stabilize38 the expression of the GalS gene.

The GalE I1-FFL is embedded in the cell within a number of additional interactions (Figure 2(b)), including regulation by the repressor GalR (which does not appear to be itself regulated35); the degradation of the inducer galactose by the galETK genes; inhibition of galactose pumps by glucose (known as inducer exclusion) and induction of galactose pumps by galactose, which act to increase inducer levels in the cell.

Here, we focus on the dynamic behavior of the galE promoter, as a representative I1-FFL system. We used a green fluorescent protein (GFP) reporter system to obtain high-resolution dynamical expression measurements of the gal system in living E. coli cells, and compared the dynamics to various control systems, such as the lac system (Figure 2(c)) that does not display the FFL connectivity and mutants deleted for the gal repressor genes. We find that the galE I1-FFL circuit exhibits response-acceleration. This acceleration is abolished in mutants and conditions that disrupt the I1-FFL motif. These findings support the theoretical predictions about the function of this motif and provide a view of its design and dynamics in a natural system.

Section snippets

Incoherent type-1 FFLs in the transcription networks of both E. coli and yeast

Transcription regulation databases of E. coli have increased in scope since the original study of the FFL types.18 Recently, Ma et al.21 have extended the FFL counts by using updated databases including EcoCyc and RegulonDB, as well as the data described by Shen-Orr et al.10 They counted the cases of FFL using genes as nodes, and reported several hundred new FFLs, including many I1-FFLs.

We repeated this counting, but defined each node in the network as an operon (a set of genes transcribed on

Discussion

This study examined the dynamic function of the incoherent type-1 FFL network motif, using the well-studied gal system of E. coli. Using high-resolution dynamic measurements, we found that this network motif exhibits overshoot dynamics that accelerates the response time of galE gene expression upon removal of glucose. This acceleration is lost when the inducer d-fucose is added, or when the FFL is disrupted by removing the binding site of the repressor galS in the galE promoter. These findings

Plasmids and strains

Promoter regions were PCR amplified from MG1655 genomic DNA with the following start and end genomic coordinates:45 lacZYA (365438-365669), galETK (791278-790262), galS (2239688-2238648). This included the entire region between open reading frames (ORFs) with an additional 50–150 bp into each of the flanking ORFs. Each promoter region was sub-cloned into XhoI and BamHI sites upstream of a promoterless gfpmut246 gene in a low copy pSC101-origin plasmid as described,41 to create a separate

Acknowledgements

We thank O. Barad and all members of our laboratory for discussions. We thank S. Adhya for the gift of strains. We thank NIH, HFSP, Minerva, ISF and the Kahn Fund for systems biology at the Weizmann Inst. of Science, for support.

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