Rational design of a bacterial transcriptional cascade for amplifying geneexpression capacity

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits wit h novel properties, Here we describe the design of a transcriptional regula tory cascade to amplify the cell response to a given signal, We used the sa licylate-responsive activators of Pseudomonas putida NahR of the naphthalen e degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and Pm. Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effecters for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on t he Pm promoter, amplifying the gene expression capacity by at least one ord er of magnitude with respect to the individual systems. By changing the hie rarchy of regulators, we showed that the specific features of the downstrea m regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplify ing system to other molecular signals.