Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

Abstract

Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant 10 homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost 11 of disrupting nitrogen uptake and assimilation.Step 2 is a switch-of-function mutation 12 that redirects NtrC away from nitrogen uptake and towards its novel function as a 13 flagellar regulator. Our results demonstrate that natural selection can rapidly rewire 14 regulatory networks in very few, repeatable mutational steps. 25Here we monitor the recovery of microbial populations from a catastrophic gene 26 deletion: bacteria engineered to lack a particular function are exposed to environments 27 that impose strong selection to re-evolve it, sometimes by recruitment of new genes to 28 regulatory networks (6, 7, 8, 9). 29In the plant-associated soil bacterium P. fluorescens, the master regulator of The starting P. fluorescens strain is AR2; this strain lacks flagella, due to deletion of 33 fleQ, and is unable to move by spreading motility due to mutation of viscosin synthase 34 (viscB), resulting in a distinctive, point-like colony morphology on spreading motility 35 medium (SMM) (12) ( Figure 1A). We grew replicate populations of AR2 on SMM; when 36 local nutrients became depleted, starvation imposed strong selection to re-evolve 37 motility. To demonstrate that this finding was not strain-specific, these experiments were 38 replicated in a different strain of P. fluorescens, Pf0-2x. This strain is a ∆fleQ variant of 39 Pf0-1, already viscosin-deficient, and is thus unable to move by spreading or swimming 40 motility. 41After 96 hours incubation of AR2 and Pf0-2x at room temperature on SMM, two 42 breakout mutations were visible conferring first slow (AR2S and Pf0-2xS) and then fast 43 (AR2F and Pf0-2xF) spreading over the agar surface (Fig. 1A). The AR2F strain 44 produces flagella, but we could not detect flagella in EM samples for AR2S (Fig. 1B). Table S1). The expression of 55 genes required for flagellum biosynthesis and chemotaxis was abolished in AR2 56 compared to wild-type SBW25 (Fig. 2A). The ntrB mutation in AR2S partially restores 57 the expression of flagellar genes, and over-activates the expression of genes involved 58 in nitrogen regulation, uptake and metabolism. The subsequent ntrC mutation in AR2F 59 reduces the expression of nitrogen uptake and metabolism genes, while further up-60 regulating flagellar and chemotaxis gene expression to wild-type levels (Fig. 2B). While 61 AR2S and AR2F showed higher growth rates than the ancestor in LB medium (the 62 medium on which the mutants arose; Tukey-Kramer HSD test, growth in LB compared 63 to AR2: AR2S, P < 0.001; AR2F, P < 0.001) (Fig. 1C), both mutants grew poorly in 64 minimal medium with ammonium as the sole nitrogen source (Tukey-Kramer HSD test, 65 growth in M9 + ammonium compared to AR2: AR2S, P < 0.001; AR2F, P = 0.001). This 66 is likely to be the result of ammonium toxicity due to the strong up-regulation of genes 67 5 involved in ammonium uptake and assimilation, ind...

A protein constructed de novo enables cell growth by altering gene regulation

“…When presented with environmental challenges, organisms adapt by using a range of strategies, including new enzymatic functions and novel regulatory processes. In natural systems, there are numerous precedents showing that selecting for growth under specified conditions can yield mutations that rewire gene regulation (27)(28)(29)(30)(31). For example, Taylor et al (27) showed that immotile mutants of Pseudomonas fluorescens subjected to selections for motility evolve by repurposing a protein that normally functions in nitrogen uptake toward a new function involving flagellar regulation.…”

Small RNAs in Bacteria and Archaea

“…If the generated RNA has an adaptive phenotypic effect by matching a target, selection may fix this regulatory pair. In comparison, evolving a new TF may be more difficult (however, see Taylor et al, 2015). Both bacterial sRNAs and eukaryotic miRNAs indeed often show signs of being recently "invented" (Cuperus, Fahlgren, & Carrington, 2011).…”

Experimental evolution can unravel the complex causes of natural selection in clinical infections

“…Remarkably, bacteria are capable of evolving new functions over a weekend, as shown in a recent study of P. fluorescens where engineered immotile strains re-evolved flagellar motility in v96 h. Starvation on solid agar imposed strong selection for motility in the immotile bacteria which responded by evolutionary rewiring of their nitrogen gene regulatory network, switching its specificity towards regulation of flagellar genes instead, thereby allowing the cells to regain the ability to build flagella and swim towards nutrients (Taylor et al, 2015). My own experimental evolution studies of bacterial populations reveal the potential for similarly rapid evolutionary gain or loss of a wide variety of ecologically important traits, including colony morphology (Brockhurst et al, 2004(Brockhurst et al, , 2005(Brockhurst et al, , 2007bVogwill et al, 2011), biofilm production (Brockhurst et al, 2007a), resistance to drugs (Habets & Brockhurst, 2012) or phages (Brockhurst et al, 2003(Brockhurst et al, , 2005, motility (Brockhurst et al, 2005;Taylor et al, 2015) and loss of social secretions such as siderophores (Brockhurst et al, 2008).…”