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

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.

Figure 1

(A) The type-1 coherent FFL (Mangan and Alon, 2003). In many cases, Y regulates its own production as shown. (B) The SUM-FFL in the flagella class 2 regulation network. X is flhDC, Y is fliA and Z is the fliLMNOPQR operon (termed fliL) and other class 2 operons. In this circuit, the activator X regulates Y, X and Y act additively to activate the output gene Z. The input Sx is the production rate of X (or, more generally, a stimulus that activates X). The input Sy regulates the activity of Y. In the flagella system, Y positively regulates its own production. (C) A more detailed view of the flagella network and the basal-body checkpoint. The flhDC promoter is controlled by several transcription factors responsive to environmental stress and starvation. The class 2 genes encode the structural proteins that make up the basal bodies. FliA is involved in a positive feedback loop called the basal body checkpoint. In this loop, the activity of FliA as a transcription factor is inhibited by binding the protein FlgM (dashed -∣ sign indicating inhibition). FlgM is exported out of the cell once the first active basal bodies are formed, by a specific transport mechanism that exports FlgM through the basal bodies (dashed inhibition symbol between the basal body and FlgM). Thus, FliA helps activate genes that produce basal bodies, which export the inhibitor FlgM out of the cell, relieving the inhibition of FliA.

Figure 2

Experimental dynamics of Z (fliL) expression in a strain containing Y (fliA, strain U306+pJM45+pJM35, RP437ΔflhD, •) and a strain deleted for Y (U307+pJM45+pJM35, RP437ΔflhDΔfliA, □) (strains and plasmids were described in Kalir and Alon, 2004).(A) Production of X (FlhDC) regulated by the araBAD promoter on a low-copy plasmid was controlled by an inducer externally added to the cells (L-arabinose). The anti-inducer D-fucose allowed deactivation of X expression. (B) Dynamics of Z expression following induction of X. Cells were grown in defined glycerol medium as described (Kalir and Alon, 2004) with saturating inducer (2 mM arabinose), and Z production rate was monitored using GFP controlled by the Z promoter. GFP fluorescence divided by cell density (OD), normalized to a maximum of one, is shown. (C) Dynamics following turn-OFF of X production. Cells were grown with inducer for 3 h and then shifted to medium with no inducer and saturating anti-inducer (50 mM D-fucose).

Figure 3

Experimental dynamics of Z (fliL) expression in wild-type cells (U16+pJM35, RP437 •), and in cells deleted for fliA (U309+pJM35, RP437ΔfliA □). Cells were grown in defined glycerol medium (Kalir and Alon, 2004) and Z production rate was monitored using GFP controlled by the Z promoter. Promoter activity, defined (Kalir and Alon, 2004) as the rate of change of GFP flourescence divided by cell density (OD), normalized to a maximum of one, is shown.