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...

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work, we reveal that a near-deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observe this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determine that this evolutionary disparity is owed to just 6 synonymous variations within the ntrB locus, which we demonstrate by swapping the sites and observing that we are able to both break (>95% to 0%) and build (0% to 80%) a deterministic mutational hotspot. Our work reveals a key role for silent genetic variation in determining adaptive outcomes.

Plant-associated bacteria face multiple selection pressures within their environments and have evolved countless adaptations that both depend on and shape bacterial phenotype and their interaction with plant hosts. Explaining bacterial adaptation and evolution therefore requires considering each of these forces independently as well as their interactions. In this review, we examine how bacteriophage viruses (phages) can alter the ecology and evolution of plant-associated bacterial populations and communities. This includes influencing a bacterial population's response to both abiotic and biotic selection pressures and altering ecological interactions within the microbiome and between the bacteria and host plant. We outline specific ways in which phages can alter bacterial phenotype and discuss when and how this might impact plant-microbe interactions, including for plant pathogens. Finally, we highlight key open questions in phage-bacteria-plant research and offer suggestions for future study.

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phagemediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.