Evolution of gene regulatory networks in a bacterial experimental system: Identifying general principles governing regulator rewiring from two contrasting mutational pathways

The timing and magnitude of gene expression in living cells are carefully controlled by circuits of cis- and trans-regulatory elements called gene regulatory networks. Components of these networks can interact with non-cognate sites (i.e. they can act promiscuously), providing an evolutionary raw-material for repurposing or rewiring regulators to take on new functions. Here we investigate two distinct evolutionary pathways for repurposing transcription factors in a bacterial experimental system. A strain of the soil bacterium Pseudomonas fluorescens lacking the flagellum master regulator FleQ is challenged to rescue flagellar expression and motility. The primary mechanism observed to rescue motility is repurposing the nitrogen assimilation regulator NtrC, which occurs reliably and repeatability via a two step process. Through knockout of the ntrC gene, a secondary rarer mechanism was identified. Genomic and transcriptomic data indicates motility rescue through repurposing of the putative regulator PFLU1132, as well as the alginate biosynthesis regulator AlgB. Through transcriptomic analysis of these two pathways, two contrasting approaches to rescuing gene expression are observed. One involving targeted rewiring of the transcription factor, and the other involving global changes to gene expression to facilitate the promiscuity of several homologous regulators. Notably, we can provide empirical
evidence for general principles constraining the evolution of transcription factors, including the expression of a regulator, and the structure of the gene regulatory network it is part of. Our work highlights the dynamic and complex functionality of transcription factors within control networks, and their ability to evolve and rewire to achieve new regulatory functions.