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.
Highlights
The mechanisms that bacteria use to transduce external stimuli into specific responses rely on connected transcriptional factors that shape circuit-like input/output devices [1]
To have a reliable test system for comparing the inputs and outputs associated with this node, we engineered these components in a strain of P. putida bearing a transcriptional Pu-luxCDABE fusion inserted into its chromosome via a mini-transposon vector (P. putida Pu?LUX, Figure 2c)
Prokaryotic regulatory systems have been used in the construction of bacterial strains with biotechnological applications, such as whole-cell biosensors to detect environmental pollutants [15,41,43,44]
Summary
The mechanisms that bacteria use to transduce external stimuli into specific responses rely on connected transcriptional factors that shape circuit-like input/output devices [1]. Such networks are comprised of interacting molecular components and can adopt different topologies [2]. XylR is optimally activated by the primary substrates of the TOL system, such as m-xylene or toluene This TF is not entirely specific for these effectors, as it responds to a large number of structural analogs. The existing characteristics of the XylR-based regulatory network have likely evolved for adjusting the tradeoff between transcriptional efficiency and physiological burden in the natural context, constraining the
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