Fig. 2: V. cholerae 3D biofilm formation is induced by cell lysate.

a, V. cholerae forms biofilms during exposure to active Vibriophage N4 (10⁶ p.f.u. ml⁻¹; see also Fig. 1d) but not when phages were inactivated by heat treatment (65 °C for 15 min). Biofilm formation is quantified as the 3D biofilm biovolume fraction (biovolume with height H > 3 µm divided by the total biovolume). Measurements were performed after 8 h of exposure to active or inactive virions. b, Following 60 min of phage exposure in liquid shaking culture with MOI = 1, the bacterial colony-forming unit count dropped substantially for the WT due to phage-induced lysis, whereas the ∆trxA mutant was unaffected by phage exposure. c, The ∆trxA mutant did not form 3D biofilms after 8 h of phage exposure (10⁶ p.f.u. ml⁻¹). The WT or ∆trxA mutant did not form 3D biofilms without phage exposure. d, Both the WT and ∆trxA mutant formed biofilms when exposed to a lysate obtained by sonication of WT V. cholerae cells (10¹⁰ lysed cells per ml) for 3 h. e, Quantification of the total biovolume (grey) and biovolume with height H > 3 µm, measured in the presence (blue, left panel) or absence (black, right panel) of a lysate obtained by sonication of WT V. cholerae cells (10¹⁰ lysed cells per ml). Confocal microscopy images show V. cholerae cells expressing sfGFP (displayed in yellow) after 3 h in the lysate-exposed or unexposed condition. f, Exposure to increasing concentrations of V. cholerae lysate solutions (obtained by sonication) resulted in a higher 3D biofilm biovolume fraction. In all panels (a–f), bars are mean values of n = 3 independent biological replicates, circles indicate individual measurements and error bars indicate the standard deviation. Statistical significances were calculated between the groups indicated by black lines in panels a–d using a two-sided Student’s t-test or relative to the unexposed condition in panel f using a one-way ANOVA with Bonferroni’s correction. Statistical results are given as exact P values in brackets in the graphs, or indicated using the following: NS, not significant (P > 0.99, unless specified otherwise in the figure), ****P < 0.0001.

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