Widening functional boundaries of the σ54 promoter Pu of Pseudomonas putida by defeating extant physiological constraints

Abstract

The extant layout of the σ⁵⁴ promoter Pu, harboured by the catabolic TOL plasmid, pWW0, of Pseudomonas putida is one of the most complex instances of endogenous and exogenous signal integration known in the prokaryotic domain. In this regulatory system, all signal inputs are eventually translated into occupation of the promoter sequence by either of two necessary components: the m-xylene responsive transcriptional factor XylR and the σ⁵⁴ containing form of RNA polymerase. Modelling of these components indicated that the Pu promoter could be upgraded to respond with much greater capacity to aromatic inducers by artificially increasing the endogenous levels of both XylR and the σ⁵⁴ sigma factor, either separately or together. To explore these scenarios, expression of rpoN, the gene encoding σ⁵⁴, was placed under the control of an orthogonal regulatory system that was inducible by salicylic acid. We generated a knock-in P. putida strain containing this construct alongside the xylR/Pu regulatory module in its native configuration, and furthermore, a second strain where xylR expression was under the control of an engineered positive-feedback loop. These interventions allowed us to dramatically increase the transcriptional capacity (i.e. absolute promoter output) of Pu far beyond its natural scope. In addition, they resulted in a new regulatory device displaying more sensitive and ultra-fast responses to m-xylene. To our knowledge, this is the first time that the working regime of a promoter has been rationally modified by releasing the constraints imposed by its innate constituents.