Fig. 3: Reduced intracellular c-di-GMP levels increase flagellar motility, decrease MSHA surface piliation, and increase frequency of MSHA retraction.

a c-di-GMP levels as quantified by LC-MS/MS. Graphs depict data as the mean, with error bars representing the standard deviation. Statistical analysis, n = 3 biological replicates per strain, one-way ANOVA compared to WT with Dunnett correction for multiple comparisons, ****p ≤ 0.0001. Source data provided as a Source Data file. b Flagellar motility in soft agar. Data presented as the ratio of mutant motility to WT motility. Graphs depict data as the mean, with error bars representing the standard deviation. n = 3 biological replicates per strain. Statistical analysis: one-way ANOVA compared to WT with Dunnett correction for multiple comparisons: Δ4DGC ****p ≤ 0.0001, Δ2PDE ***p = 0.0002, ΔpilT *p = 0.0141. Source data provided as a Source Data file. c Measurement of c-di-GMP levels in single cells inside a flow cell using the biosensor. Lines indicate the median RFI values per time point, and the shaded areas represent the 95% confidence intervals obtained from the bootstrap sampling distribution of the median RFI values. Time 0 H corresponds roughly to when cells initially encounter the surface of the flow cell. (d) Plot of the probability of c-di-GMP increase, calculated by comparing the bootstrap sampling distributions of the median RFI values between every time point (I_t) and the first (I₁). C-di-GMP is determined to be increasing if I_t/I₁ ≥ 1.05. e Analysis of surface MSHA production via hemagglutination (HA) assay. The reciprocal of the lowest fold dilution at which equivalent cell levels were able to agglutinate sheep erythrocytes (HA Titer) is plotted as the mean with error bars representing the SEM. For each strain n = 5 biological replicates were analyzed, with two technical replicates performed for each biological replicate. WT value is the same as shown in Fig. 2f. ND no observable hemagglutination at the highest cell concentration. Statistical analysis: each mutant HA titer was compared to WT via unpaired two-tailed Student’s t-Test, ****p ≤ 0.0001. Source data provided as a Source Data file. f Representative overlay images of filtered-fluorescence and phase-contrast channels of mshA^T70C strains within surface-associated cells stained with AlexaFluor 488 C₅ maleimide dye. Images representative of three independent analyses. Scale bars = 2μm. g VGJΦ phage transduction levels. All in mshA^T70C strain. Individual data points of kanamycin-resistant CFU/mL plotted with line at the mean and error bars representing the standard deviation. Biological replicates: n = 8 per strain. Statistical analysis: unpaired two-tailed Student’s t-Test, ****p ≤ 0.0001. Source data provided as a Source Data file.