Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25

Abstract

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these-a nonsynonymous point mutation in the housekeeping sigma factor rpoD-is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB. A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression. We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosomal gene expression, and ultimately an increase in CAP expression.

Keywords: evolution; genetics; microbiology.

Fig. 1.

Emergence of colony switching in reverse evolution experiment (REE) Line 1. (A) Cartoon of one Line of the 12-Line REE (Beaumont et al. 2009). Populations were subjected to bouts of selection in shaken (environment A) or static (environment B) liquid KB. After each bout, cells were plated on KB agar and a colony with novel morphology was purified and used to start the next round in the opposite environment. (B) Line 1 phenotypes and genotypes. Each Line 1 strain produces colonies distinct from those of its immediate ancestor (first row). Strains differ in their ability to produce cellulose (cells grown on KB+calcofluor agar; second row). Mutations are noted as “gene (mutation)” at the point of fixation. (C) Bi-directional colony switching in 1B⁴. 1A⁴ generates colonies of a single type (top), whereas 1B⁴-Tr (middle) or -Op colonies (bottom) generate a mixture. (D) 1B⁴ cells are capsulated (Cap⁺) or noncapsulated (Cap⁻); 1A⁴ cells are generally Cap⁻. (E) SBW25 colonies are uniform whereas SBW25-carB* (SBW25 into which the c2020t carB mutation is engineered) shows colony bistability. Colonies grown on KB agar (28 °C, 48 h); cells grown in shaken KB (16–24 h) before staining and bright field or fluorescence microscopy. Exposure of some images altered in Preview. Tr, translucent; Op, opaque.

Fig. 2.

Emergence of colony switching in REE Line 6. (A) Phenotypes and genotypes of Line 6: colony morphology on KB agar (row 1), morphology of cells grown in shaken KB microcosms and stained with India ink (row 2), ability of cells grown on KB agar with calcofluor to produce cellulose (row 3), and mutations (shown at point of occurrence; bottom). (B) Colony and cell morphologies of 6B⁴ and its nonswitching immediate ancestor, 6A⁴. 6B⁴ gives rise to translucent (Tr) and opaque (Op) colonies, plus capsulated and noncapsulated cells. (C) The proportion of capsulated cells in various populations during stationary phase. Each bar is the mean of five replicate populations grown overnight in KB microcosms. Error bars are one standard error and stars denote statistical significance (***P < 0.001). Contrast and/or exposure of some images altered in Preview.

Fig. 3.

The t1682c rpoD mutation causes the emergence of capsule switching in both the presence and absence of the other Line 6 mutations. (A) Engineered strains carrying the rpoD mutation develop a mixture of Tr/Op colonies on KB agar after 48 h, and a mixture of Cap⁺/Cap⁻ cells (cells grown overnight in KB glass microcosms and stained with India ink before bright field microscopy). Saturation and brightness of some photographs altered in Preview. (B) The proportion of capsulated cells in populations of various genotypes during stationary phase. Each bar represents the mean of five replicate populations grown overnight in KB glass microcosms. Error bars represent one standard error and stars show statistical significance (**0.01 < P < 0.001).

Fig. 4.

Three rpoD mutations have different effects on capsulation and growth. Three amino acid substitutions leading to switching have been identified in σ⁷⁰. Each of these changes occurs in region 4, which encodes an H–T–H motif that binds to the −35 consensus sequence of σ⁷⁰-dependent promoters.Nonessential region (A). In each case, the amino acid substitution leads to the emergence of two colony phenotypes or sectored colonies (B; colonies grown on KB agar for ∼56 h) and an increase in capsulation during a capsule counting assay in stationary phase (C; bars are the mean of five replicates). (D) Twenty-four-hour growth curves in shaken KB medium at 28 °C. Measurements were taken every 5 min, with eight replicates for each strain (against KB blanks). Mean maximum growth rates (E) and lag times (F) were calculated using a sliding window of six data points. Error bars on all graphs how one standard error.

Fig. 5.

The fitness effect of switch-causing rpoD and carB mutations is lineage dependent. (A) The t1682c rpoD and c2020t carB mutations both cause colony switching in the 6A⁴ and 1A⁴ backgrounds (seen as two colony types and/or sectors on KB agar, 72 h). (B) A capsule counting assay in stationary phase shows that the rpoD and carB mutations cause an increase in capsulation relative to the immediate ancestor in both lineages (Wilcoxon rank sum tests P = 0.02857*). Bars = mean of four replicates, error bars are one SE. (C) Box plots of the fitness of the evolved type versus the engineered type in Line 6 and Line 1. Competition assays (1:1) were performed under the REE conditions in which the evolved types were originally isolated (72-h static microcosms). Values greater than 1 indicate a higher relative fitness of the first competitor (evolved types). Both competitions show a significant deviation from 1 (one-sided one sample t-test *P < 0.05, ***P < 0.001).

Fig. 6.

The ribosome–RsmAE model of capsule switching in 6B⁴. (A) As perRemigi et al. (2019), relative pools of three molecules determine Cap state: ribosomes (gray mushrooms), RsmAE (purple circles), pflu3655 mRNA. Initially, ribosomes and RsmAE compete for binding sites on pflu3655 mRNA. If ribosomes bind then translation follows, giving Pflu3655 (red diamond) and CAP synthesis. Pflu3655 forms a positive feedback loop (pflu3655 transcription) that maintains the Cap⁺ state. Change to Cap⁻ requires RsmAE to outcompete the other components (reduction of pflu3655 mRNA and/or production of RsmA/E). Intracellular components are predicted to influence Cap positively (black boxes) or negatively (purple boxes) by altering relative pools. Solid outlines = components with supporting evidence, dotted lines = untested. (B) Model of RsmAE function in Pseudomonas fluorescens SBW25. RsmAE binds to RNA sequences found in short RNAs (rsmYZ; right) and various promoters (left). The net binding of RsmAE to a promoter affects translation of the mRNA through competitive binding with other translational machinery (e.g., ribosomes). (C) rsmA1 deletion in 6B⁴ increases Cap⁺ in exponential phase (two sample t-test P = 1.112 × 10⁻³). (D) mvaT (pflu4939) deletion in 6B⁴ or 1B⁴ increases Cap⁺ in exponential phase (Wilcoxon test P = 0.009761; two sample t-test P = 1.952 ×10⁻¹⁵). (E) Nonpolar insertions in pflu3656, pflu3657, RsmAE regulators (gacA, gacS), and translation machinery (truA, gidA, thiI) reduce 6B⁴ capsulation in exponential phase. Bars = mean of 5 (B, C) or 3 (D) replicates, error bars 1 SE.