Adaptive and maladaptive consequences of deregulation in a bacterial gene regulatory network

The archetypal PhoQP two-component system from Enterobacteria regulates crucial pathways like magnesium homeostasis in Escherichia coli and virulence factor expression in Salmonella enterica. Previously we had reported that a laboratory strain of E. coli rapidly accumulated loss-of-function mutations in the mgrB gene, a negative feedback regulator of PhoQP, when evolved in the presence of the antibiotic trimethoprim. Hyperactive PhoQP enhanced the expression of dihydrofolate reductase (folA), target of trimethoprim, resulting in antibiotic tolerance. Here we ask, firstly, how important are mutations in mgrB for trimethoprim resistance? Using laboratory evolution, we show that trimethoprim resistance evolves by different mutational trajectories under condition of high and low PhoQP activity. Mutations in mgrB are only fixed when PhoQP is active. Importantly, loss of functional MgrB, though itself only mildly beneficial, enhances the fixation probability of trimethoprim-resistant bacteria under selection and this can be explained by epistasis between mgrB and folA loci. As a result, the activation status of PhoQP directly impacts how fast resistance is acquired by evolving populations of E. coli. Secondly, we investigate why negative feedback may be needed in the PhoQP system. We show that under drug-free conditions MgrB is required to mitigate the fitness costs of pervasive gene dysregulation by hyperactive PhoQP. Using RNA-seq transcriptomics and genetic analyses, we demonstrate that PhoQP-hyperactivation perturbs the balance of RpoS and RpoD-regulated transcriptional programs, and spontaneous mutations in rpoS rectify this imbalance. We propose that deregulation can be adaptive or maladaptive depending on the environmental context and this explain the evolution of negative feedback in bacterial gene regulatory networks.