Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

Abstract

Prokaryotic transcription factors (TFs) that bind small xenobiotic molecules (e.g., TFs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. XylR, the master TF for expression of the m-xylene biodegradation operons encoded in the TOL plasmid pWW0 of Pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbenzylalcohol (3MBA). We have examined whether such a relaxed regulatory scenario can be reshaped into a high-capacity/high-specificity regime by changing the connectivity of this effector-sensing TF within the rest of the circuit rather than modifying XylR structure itself. To this end, the natural negative feedback loop that operates on xylR transcription was modified with a translational attenuator that brings down the response to 3MBA while maintaining the transcriptional output induced by m-xylene (as measured with a luxCDABE reporter system). XylR expression was then subject to a positive feedback loop in which the TF was transcribed from its own target promoters, each known to hold different input/output transfer functions. In the first case ( xylR under the strong promoter of the upper TOL operon, Pu), the reporter system displayed an increased transcriptional capacity in the resulting network for both the optimal and the suboptimal XylR effectors. In contrast, when xylR was expressed under the weaker Ps promoter, the resulting circuit unmistakably discriminated m-xylene from 3MBA. The non-natural connectivity engineered in the network resulted both in a higher promoter activity and also in a much-increased signal-to-background ratio. These results indicate that the working regimes of given genetic circuits can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops.

Author Summary

It is generally taken for granted that promoters regulated by transcriptional factors (TFs) that respond to small molecules control their specificity to given effectors by tightening or relaxing the intrinsic dual interaction between the TF and the particular inducer. One such promoter is Pu, which drives expression of an operon for the biodegradation of m-xylene by the soil bacterium P. putida mt-2. While XylR, the chief TF of this system, binds this substrate and activates Pu, the same regulator responds, to a lesser extent, to 3-methylbenzylalcohol and thus also activates the promoter. This work provides evidence that such natural effector promiscuity of the system can be altogether suppressed by replacing the naturally occurring negative autoregulation loop that governs XylR expression with an equivalent positive feedback loop. Based on this result, we argue that signal specificity of a given regulatory device depends not only on the TF involved but also on TF connectivity to upstream signals and downstream targets.