Fig. 5

Fig. 5

Figure 5. SpdE regulates Aer01 chemokinesis and biofilm formation via modulation of c-di-GMP

(A) Intracellular quantification of c-di-GMP in the absence or presence (1 mM) of SpdE ligands. WT^anc, ancestral Aer01; ΔsdE, knockout strain; ΔspdE_comp and ΔspdE_comp^evol, ΔspdE complemented with a WT copy of spdE or an evolved mutant allele, respectively. n = 3 independent cultures.

(B) Population-level Aer01 motility for WT^anc and ΔspdE determined by exploration assay (see Figure S7); “reference” strain/condition specified at bottom. n = 2–6 experimental replicates. Bars are mean ± SD. Dotted line represents fold change of 1 (i.e., no difference in motility). Blue outlined bars, ΔspdE; black outlined bars, WT^anc.

(C) Representative mass projection plots of movies of fluorescently tagged WT^anc to visualize motility. White trails are “swimming tracks” of motile cells. Mass projections of all movies shown in Figure S8.

(D) Quantification of number of motile cells from all movies (see Figure S8) of WT^anc across a range of ligand. Mean ± SEM is plotted. n = 3 movies/condition.

(E) Swim velocities of individual Aer01 motile cells (WT^anc and ΔspdE) incubated ± 1 mM proline. Each box represents data from a single movie (n = 3–4 movies per strain/condition; number of motile cells/movie indicated below each plot); black outline, WT^anc; blue outline, ΔspdE; red fill, proline condition. Statistical groups (each set of box plots for a strain/condition) were compared by combining averages for each group (ANOVA, Tukey’s range test).

(F) Quantification of biofilm formation for WT^anc and ΔspdE Aer01 ± ligand. Black outline, WT^anc; blue outline, ΔspdE. ANOVA, Tukey’s range test. n = 9, 3 experimental replicates.

(G) SpdE ligand impact on biofilm dispersal. Statistical significance (compared with buffer) determined by unpaired, two-tailed t test, ^∗p < 0.05. Pro, proline; Val, valine; Gly, glycine. n = 9–12; 3–4 experimental replicates.

(H) Chemotaxis response of Aer01 strains to amino acids. All amino acid responses compared with buffer control. Each data point represents an independent experiment. Bars represent mean ± SD. Dotted line represents fold change of 1 (i.e., no difference in chemotaxis); n = 3–4 experimental replicates.

(I) Competitive indices of WT^anc, ΔspdE, ΔcheA, and ΔspdE/ΔcheA strains when competed in larval fish against a WT reference strain (see Figure S1A). Dotted line indicates CI of 1 (i.e., no competitive advantage). n ≥ 8 fish/condition, one experimental replicate. Data for WT^anc and ΔspdE are the same as those plotted in Figure 2A. ^∗p < 0.05, two-tailed t test performed on log-transformed CI data. For box and whisker plots in (E), (F), and (G), boxes represent median and interquartile ranges; whiskers represent the min and max. See also Figures S5 and S6.