Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix

Abstract

Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains.

Fig. 1.

Biofilm expansion is a collective mechanism based on extracellular matrix production. (A) Top view of B. subtilis biofilm morphology and expansion on MSgg agar plates at different timepoints for wild-type (WT) strain 3610 (top row), the flagella mutant hag (mid row), and the eps mutant (bottom row). Scale bar, 1 mm. A central spot is apparent in all the pictures and represents the initial inoculum of cells. (B) Radial growth obtained by averaging every two hours over 25 colonies for each of the three strains WT (black squares), hag (blue open circles), and eps (red dots); shades indicate the standard deviation. The eps mutant shows a severely limited horizontal expansion. In these conditions, flagella do not play a significant role in the horizontal expansion of the biofilm. (C) Growth curves of the three strains: WT, hag and eps in shaking liquid culture. (D) Consecutive frames from a time-lapse high resolution movie of the edge of the growing wild-type biofilm. The marker indicates the position of a particular cell that slides on top of the agar during expansion.

Fig. 2.

A model of osmotically driven growth predicts a transition between an initial vertical swelling and a later horizontal expansion, as illustrated by simulations and asymptotics of the model equations. The single nondimensional parameter K controls the transition. (A) Simulated profiles for K = 10^-5 at different times color coded from green at tg = 0 to red at tg = 5. The edge of the biofilm is marked on each profile through the slope . (B) Radius of the biofilm defined as the point where the profile is maximally steep; the black circles represent the results of simulations of Eq. 5 in two planar dimensions, for different values of K from 10^-1 to 10^-5; the blue lines are the asymptotic scalings given by the self-similar solution 7–9 modified for planar coordinates (see Materials and Methods). (C) The slope at the edge of the colony initially increases as the biofilm swells vertically as indicated by the arrow. After reaching the critical value , it decreases as the biofilm expands and smoothens; the maximum (critical) slope defines the transition time (symbols as in B). (D) Critical slope and (E) time of transition as a function of K (symbols as in B).

Fig. 3.

Quantitative comparison with experimental data of biofilm profiles shows that the wild type undergoes the predicted swelling/spreading transition, whereas the eps mutant does not. Biofilm shape at intervals of 1.5 h for WT (A) and eps mutant (B), obtained by azimuthally averaging the logarithm of the transmitted light intensity (see Materials and Methods and SI Text). The profiles are shown in units of the initial height h₀ defined as the average height in the central plateau of the biofilm at time 0; we show the time evolution color-coded from green at t = 1.5 h to dark green (WT) and red (eps) at t = 18 h. (C) Volume of the biofilm as a function of time, calculated from the profiles in A and B. We obtain the average doubling time by fitting the wild-type growth curve with a single exponential giving g^-1 ∼ 2.3 h. (D) The maximum slope of the wild-type biofilm (black squares) first increases when the biofilm swells vertically and then decreases as the biofilm spreads horizontally and smoothens out. Values are shown in units of the initial value. Time is nondimensionalized with g^-1 = 2.3 h obtained from the previous fit. The experimental points closely follow the theoretical prediction 9 with the single fitting parameters K ∼ (1 ± 0.2) × 10^-5. The eps mutant (red circles) continues to steepen, with no apparent shape transition. (E) Wild-type biofilm radius (black squares) starts to expand after a critical time; eps biofilms (red circles) do not display the transition. Data for the wild type are in good agreement with the prediction 8. The radius is defined as the point of maximum slope and it is computed from the profiles. Error bars in D and E are defined as described in Materials and Methods. (F) Average horizontal extension of hag (blue) and hag eps (red) biofilms after 30 h from plating as a function of agar concentration. The error bar is the standard deviation computed over three replicas. Data are obtained with the protocol described in Radial growth.