A coherent feed‐forward loop with a SUM input function prolongs flagella expression in Escherichia coli

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Abstract
Complex gene‐regulation networks are made of simple recurring gene circuits called network motifs. The functions of several network motifs have recently been studied experimentally, including the coherent feed‐forward loop (FFL) with an AND input function that acts as a sign‐sensitive delay element. Here, we study the function of the coherent FFL with a sum input function (SUM‐FFL). We analyze the dynamics of this motif by means of high‐resolution expression measurements in the flagella gene‐regulation network, the system that allows Escherichia coli to swim. In this system, the master regulator FlhDC activates a second regulator, FliA, and both activate in an additive fashion the operons that produce the flagella motor. We find that this motif prolongs flagella expression following deactivation of the master regulator, protecting flagella production from transient loss of input signal. Thus, in contrast to the AND‐FFL that shows a delay following signal activation, the SUM‐FFL shows delays after signal deactivation. The SUM‐FFL in this system works as theoretically predicted despite being embedded in at least two additional feedback loops. The present function might be carried out by the SUM‐FFL in systems found across organisms. ### Synopsis Under unfavorable growth conditions, bacteria such as Escherichia coli synthesize multiple flagella, which allow them to swim away towards a ‘better life’. The flagellum is a highly organized and complex structure, which requires the correct assembly of a number of proteins, notably the motor complex in the cytoplasmic membrane. Biosynthesis of the motor components is subject to a tight control through genetic regulatory circuits. In these circuits, the environmental situation is ‘sensed’ by the bacterium which regulates the activity of certain transcription factors accordingly such that they dynamically regulate the appropriate production rate of flagella proteins. In general, complex gene regulation networks are built of a combination of simple gene circuits, also called network motifs. It is important to understand the function of each motif in order to build a complete dictionary of basic circuit elements and their functions. [Figure 1][1] illustrates the structure of one of the most common network motifs, the feed‐forward loop (FFL). In such a circuit transcription factor X (i.e. master flagella activator FlhDC) activates a second transcription factor Y (FliA), and both activate the output genes Z that build the flagella motor. If the activators X and Y operate in an additive fashion to regulate the genes Z, one can specify the FFL circuit as an FFL with a SUM input function (SUM‐FFL). This network motif is the focus of our paper. We employed flagella biosynthesis as a model system and analyzed the dynamics of the SUM‐FFL network motif using high‐resolution gene expression measurements in living cells based on green‐fluorescent protein reporters. We found that this network motif prolongs flagella expression even when the master regulator X is deactivated. Flagella production is prolonged for a time period that is of the same order of magnitude as the one required to assemble a flagellum. The SUM‐FFL motif therefore seems to protect flagella synthesis from transient loss of input signal. What is the need for such a protection mechanism? It is known that environmental signals such as carbon starvation, temperature, etc. govern the synthesis of the master regulator. As these environmental factors fluctuate as may occur, for example, when bacteria swim from place to place, the SUM‐FFL motif makes the flagella biosynthesis system insensitive to brief periods in which the master regulator X is deactivated. The protection function we assigned to the SUM‐FFL motif characterized here might be extended to other biological systems as well. This paper is the first experimental study of the function of a common network motif, one of the main building blocks of gene regulation networks. This gene circuit is shown to protect gene systems from transient loss of input signal. Mol Syst Biol. 1: 2005.0006 [1]: #F1