Bet hedging-stochastic switching between phenotypic states-is a canonical example of an evolutionary adaptation that facilitates persistence in the face of fluctuating environmental conditions. Although bet hedging is found in organisms ranging from bacteria to humans, direct evidence for an adaptive origin of this behaviour is lacking. Here we report the de novo evolution of bet hedging in experimental bacterial populations. Bacteria were subjected to an environment that continually favoured new phenotypic states. Initially, our regime drove the successive evolution of novel phenotypes by mutation and selection; however, in two (of 12) replicates this trend was broken by the evolution of bet-hedging genotypes that persisted because of rapid stochastic phenotype switching. Genome re-sequencing of one of these switching types revealed nine mutations that distinguished it from the ancestor. The final mutation was both necessary and sufficient for rapid phenotype switching; nonetheless, the evolution of bet hedging was contingent upon earlier mutations that altered the relative fitness effect of the final mutation. These findings capture the adaptive evolution of bet hedging in the simplest of organisms, and suggest that risk-spreading strategies may have been among the earliest evolutionary solutions to life in fluctuating environments.

Biocides, such as herbicides, are routinely tested for toxicity but not for sublethal effects on microbes. Many biocides are known to induce an adaptive multiple-antibiotic resistance phenotype. This can be due to either an increase in the expression of efflux pumps, a reduced synthesis of outer membrane porins, or both. Exposures of Escherichia coli and Salmonella enterica serovar Typhimurium to commercial formulations of three herbicides—dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup)—were found to induce a changed response to antibiotics. Killing curves in the presence and absence of sublethal herbicide concentrations showed that the directions and the magnitudes of responses varied by herbicide, antibiotic, and species. When induced, MICs of antibiotics of five different classes changed up to 6-fold. In some cases the MIC increased, and in others it decreased. Herbicide concentrations needed to invoke the maximal response were above current food maximum residue levels but within application levels for all herbicides. Compounds that could cause induction had additive effects in combination. The role of soxS, an inducer of the AcrAB efflux pump, was tested in β-galactosidase assays with soxS-lacZ fusion strains of E. coli. Dicamba was a moderate inducer of the sox regulon. Growth assays with Phe-Arg β-naphtylamide (PAβN), an efflux pump inhibitor, confirmed a significant role of efflux in the increased tolerance of E. coli to chloramphenicol in the presence of dicamba and to kanamycin in the presence of glyphosate. Pathways of exposure with relevance to the health of humans, domestic animals, and critical insects are discussed.

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution.

Pseudomonas fluorescens is a model for the study of adaptive radiation. When propagated in a spatially structured environment, the bacterium rapidly diversifies into a range of niche specialist genotypes. Here we present a genetic dissection and phenotypic characterization of the fuzzy spreader (FS) morphotype-a type that arises repeatedly during the course of the P. fluorescens radiation and appears to colonize the bottom of static broth microcosms. The causal mutation is located within gene fuzY (pflu0478)-the fourth gene of the five-gene fuzVWXYZ operon. fuzY encodes a b-glycosyltransferase that is predicted to modify lipopolysaccharide (LPS) O antigens. The effect of the mutation is to cause cell flocculation. Analysis of 92 independent FS genotypes showed each to have arisen as the result of a loss-of-function mutation in fuzY, although different mutations have subtly different phenotypic and fitness effects. Mutations within fuzY were previously shown to suppress the phenotype of mat-forming wrinkly spreader (WS) types. This prompted a reinvestigation of FS niche preference. Time-lapse photography showed that FS colonizes the meniscus of broth microcosms, forming cellular rafts that, being too flimsy to form a mat, collapse to the vial bottom and then repeatably reform only to collapse. This led to a reassessment of the ecology of the P. fluorescens radiation. Finally, we show that ecological interactions between the three dominant emergent types (smooth, WS, and FS), combined with the interdependence of FS and WS on fuzY, can, at least in part, underpin an evolutionary arms race with bacteriophage SBW25F2, to which mutation in fuzY confers resistance.A DAPTIVE radiation-the rapid emergence of phenotypic and ecological diversity within an expanding lineage-is among the most striking of evolutionary phenomena (Darwin 1859;Lack 1947;Dobzhansky 1951;Simpson 1953;Schluter 2000;Kassen 2009;Losos 2010). Fueled by competition and facilitated by ecological opportunity, successive radiations have shaped much of life's diversity. Of central importance are the phenotypic innovations that fashion the fit between organism and environment (Schluter 2000).Understanding the nature of these innovations and the pathways by which they emerge and rise to prominenceoften in parallel across estranged populations experiencing similar environments-is a central issue (Colosimo et al. 2005;Bantinaki et al. 2007;Conte et al. 2012). How mutational processes generate the variation presented to selection (McDonald et al. 2009;Braendle et al. 2010), how genetic architecture underpinning extant phenotypes determines the capacity of lineages to generate new and adaptive phenotypes (Poole et al. 2003;Wagner and Zhang 2011), and how ecological factors drive phenotypic divergence (Schluter 2009) are questions of seminal interest.The relative simplicity of microbial systems, their capacity for rapid evolutionary change, and advances in technology that enable detailed genotypic traceability offer a unique opportunity to log moment-by-...