Fig. 3: Exogenous PG triggers the V. cholerae 3D biofilm formation program.

a, V. cholerae (Vc) cells grew into 3D biofilms when exposed to lysate (10¹⁰ lysed cells per ml, obtained by sonication) of V. cholerae cells (blue bar; see also Fig. 2f) or lysates of other Gram-negative species (yellow bars: Ec, E. coli; Pa, P. aeruginosa) or lysates of Gram-positive species (purple bars: Bs, B. subtilis; Sa, S. aureus). Biofilm formation was quantified as the 3D biofilm biovolume fraction after 3 h of exposure to lysate (or unexposed control). b, Different fractions of a V. cholerae lysate, obtained by filtration with different pore sizes (3–300 kDa), showed reduced biofilm induction capacity for smaller filter pore sizes. The lysate was obtained by sonicating 10¹⁰ Vc cells per ml, followed by sterilization using a 0.22 µm filter, followed by fractionation with filters of different pore sizes. c, Comparison of the biofilm induction capacity of different V. cholerae lysates: lysate of WT whole cells (blue, similar to a and Fig. 2f), lysate of cells that lacked a cell wall (spheroplasts, yellow) or cell wall fragments purified from a lysate of WT whole cells (purple), relative to the unexposed condition (black). d, Exogenously added pure PG (300 µg ml⁻¹) induced 3D biofilm formation of V. cholerae. Confocal microscopy image shows V. cholerae cells (yellow, constitutively expressing sfGFP) exposed to PG for 3 h. e, V. cholerae biofilm formation after 3 h of PG exposure increased with increasing concentration of PG. Biofilm formation was quantified as the fraction of 3D biofilm biovolume with height H > 3 µm. We estimate that a PG concentration of 300 µg ml⁻¹, solubilized by sonication, approximately corresponds to the PG concentration in lysate of 10¹⁰ lysed cells per ml. f, V. cholerae WT cells were exposed to purified V. cholerae PG (300 µg ml⁻¹ in LB) which was either undigested or treated with enzymes that cleave specific bonds in PG. The scheme illustrates which bonds are cleaved by each enzyme. g, Exposure of V. cholerae WT cells to PG (300 µg ml⁻¹) for only 5 min followed by 175 min of exposure to medium without PG, or exposure to PG for 180 min induced similar levels of 3D biofilm formation. In all panels (a–g), 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 as indicated by the black lines (in a, e and f) or relative to the lysate condition (in b) using a one-way ANOVA with Bonferroni’s correction; in panels c and g, a two-sided Student’s t-test was used. 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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