Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity

Abstract

Understanding the connections among genotype, phenotype, and fitness through evolutionary time is a central goal of evolutionary genetics. Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations and show substantial morphological and fitness differences. Previous work identified genes contributing to the evolutionary success of WS, in particular the di-guanylate cyclase response regulator, WspR. Here we scrutinize the Wsp signal transduction pathway of which WspR is the primary output component. The pathway has the hallmarks of a chemosensory pathway and genetic analyses show that regulation and function of Wsp is analogous to the Che chemotaxis pathway from Escherichia coli. Of significance is the methyltransferase (WspC) and methylesterase (WspF) whose opposing activities form an integral feedback loop that controls the activity of the kinase (WspE). Deductions based on the regulatory model suggested that mutations within wspF were a likely cause of WS. Analyses of independent WS genotypes revealed numerous simple mutations in this single open reading frame. Remarkably, different mutations have different phenotypic and fitness effects. We suggest that the negative feedback loop inherent in Wsp regulation allows the pathway to be tuned by mutation in a rheostat-like manner.

Figure 1.—

Model for the regulation and function of the Wsp pathway based on the Che (chemotaxis) pathway of enteric bacteria. The MCP (WspA), scaffold proteins (WspB and WspD), and kinase (WspE) form a membrane-bound receptor-signaling complex (IM, inner membrane; OM, outer membrane). WspR is a DGC response regulator and the primary output of the pathway. When phosphorylated, WspR catalyzes the synthesis of cyclic-di-GMP, an allosteric activator of cellulose biosynthetic enzymes. The level of kinase activity is controlled by the opposing activities of methyltransferase (WspC) and methylesterase (WspF), which add and remove (respectively) methyl groups from conserved glutamate residues on the signaling domain of the MCP (asterisks). WspC is constitutive, whereas WspF becomes active upon receipt of phosphoryl groups from the kinase. Addition of methyl groups to glutamate residues on the MCP by WspC causes the kinase to autophosphorylate and results in activation of WspF (and WspR); phosphorylated WspF removes methyl groups and thus resets the WspE kinase to an inactive state. As in the Che pathway of E. coli, the model presented here predicts that Wsp continually oscillates between active and inactive states. According to the model, a mutation in WspF that decreases WspF function (or removes functionality altogether) will result in constitutive activation of the kinase, constitutive activation of WspR, overproduction of c-di-GMP, and overproduction of cellulose and other adhesive polymers, resulting in expression of the WS phenotype.

Figure 2.—

Fitness of the 26 independently isolated wrinkly spreader mutants relative to ΔpanB-marked LSWS (hatched bar). Fitness was measured in static broth microcosms over the course of 24 hr. A fitness of 1.0 means that the fitness is identical to LSWS ΔpanB. Open bars represent strains with mutations within wspF; shaded bars represent mutants that are wild type for wspF. Error bars are means and 95% confidence intervals from four replicate assays. In a separate experiment, the fitness of a defined wspF null mutant (SM ΔwspF) was determined relative to LSWS ΔpanB: the fitness of SM ΔwspF was indistinguishable to WS_G (see text).

Figure 3.—

A model to account for the effect of wspF mutations on the signaling status of the Wsp pathway. The Wsp signaling machinery (WspA, WspB, WspD, WspE) is shown as a box with the letters C and F indicating the proteins WspC (methyltransferase) and WspF (methylesterase), respectively. The links indicate activations (arrows) or repressions (bar ends). In the absence of a stimulatory ligand, the signaling machinery oscillates between an active (solid) and inactive (open) state, depending on the combined (and opposing) effects of WspC and WspF, which cause the cell to achieve a particular balance between phosphorylated and nonphosphorylated WspR. (Left) The signaling state of the wild-type Wsp pathway that spends time in both active (upward diagonal lines) and inactive (downward diagonal lines) states. Mutations in wspF that reduce protein function, even just slightly, destroy the capacity of the pathway to fluctuate between activity states, producing instead a steady-state output (a constant level of phosphorylated WspR). The precise strength of the output signal depends on the degree to which WspF function is reduced and is maximal when WspF function is completely eliminated (right). Different mutations are predicted to tune the pathway to different output levels (middle panels).