Fig. 1: V. cholerae forms 3D biofilms following phage exposure.

a, Confocal image time series of V. cholerae (shown in yellow, constitutively expressing sfGFP) grown in a flow chamber. At time t = 0 h, cells were exposed to a continuous flow of Vibriophage N4 (phage-exposed, 10⁶ p.f.u. ml⁻¹, top row of images) or medium without phages (unexposed, bottom row). Bacterial cells that are continuously exposed to phages show an initial decrease in total biovolume due to phage infection, followed by growth as 3D biofilms. In the absence of phage exposure, cells grow as a surface-covering 2D lawn without 3D structure. b, Rendered images of phage-exposed (top) and unexposed (bottom) V. cholerae cells at t = 8 h. Cells are coloured according to their height H above the bottom surface of the microfluidic chamber. c, Quantification of the total bacterial biovolume (grey) and biovolume with height H > 3 µm over time, when the cells are exposed (blue, left panel) or unexposed (black, right panel) to Vibriophage N4 (10⁶ p.f.u. ml⁻¹). Biovolume is quantified using BiofilmQ as the volume (µm³) occupied by fluorescent bacterial cells. d, 3D biofilm formation increases with increasing phage titre. 3D biofilm formation is quantified here as the ‘biofilm biovolume fraction’, measured after 8 h. The ‘biofilm biovolume fraction’ is defined as the biovolume with height H > 3 µm, divided by the total biovolume. In panels c and d, bars are mean values of n = 3 independent biological replicates, circles indicate individual measurements and error bars indicate the standard deviation. Statistical significances in panel d were calculated relative to the unexposed condition, using a one-way ANOVA (analysis of variance) with Bonferroni’s correction; NS, not significant (P > 0.99, except for unexposed versus phage titre 10³ where P = 0.67); **P = 0.0081; ****P < 0.0001.

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