Coupling between distant biofilms and emergence of nutrient time-sharing

Abstract

Bacteria within communities can interact to organize their behavior. It has been unclear whether such interactions can extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations can become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to antiphase oscillations. This results in time-sharing behavior, where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

Fig. 1. Distant biofilms synchronize their growth dynamics

(A) Individual biofilms undergo metabolic oscillations that periodically halt growth. The metabolic oscillations are facilitated by electrical communication, which can extend beyond one biofilm to couple distant biofilms (cyan signals). In addition, two biofilms can also be coupled through competition for nutrients (red arrows). (B) Schematic depicting two biofilms grown on the two sides of a microfluidic chamber, with steady media flow. Purple and orange rectangles represent regions shown in (C). (C) Filmstrip showing the edges of a biofilm pair over time. Cyan indicates fluorescence of thioflavin T (ThT), a cationic fluorescent dye that reports membrane potential within the biofilm. Scale bar, 50 μm; h, hours. (D) Growth-rate oscillation measured by the expansion speed of biofilm edges shown in (C). (E) Membrane-potential oscillation measured from the mean ThT fluorescence at biofilm edges shown in (C).

Fig. 2. Synchronization between biofilms is governed by communication and competition

(A) Phase diagrams computed using a mathematical model of coupled phase oscillators show in-phase (green shading) and antiphase (red shading) oscillations. The colored dots indicate the experimental validations shown in the following panels. (B to D) Experimental results for wild-type (WT) (B), ΔtrkA (C), and ΔgltA (D) biofilms. For each strain, the biofilm pairs showed in-phase (phase difference of ~0) oscillations at high glutamate (glu) concentrations and antiphase (phase difference of ~p) oscillations at low glutamate concentrations. In each panel, the filmstrip shows the membrane-potential oscillation of a representative biofilm pair (scale bars, 50 μm), with corresponding time traces (color-coded by biofilm). The scatterplots show membrane potentials of biofilm pairs (n = 3 experiments per plot, one dot per time point). (E) Three-dimensional phase diagram summarizing model predictions and experimental validations. The gray-shaded surface depicts the boundary between regions of in-phase and antiphase oscillations. The black and cyan lines indicate the corresponding two-dimensional phase diagram boundaries shown in (A).

Fig. 3. Time-sharing resolves nutrient competition between biofilms

(A) Antiphase oscillations (time-sharing) allow each biofilm to take turns accessing the full quantity of supplied nutrients during its growth phase. In contrast, in-phase oscillations (resource-splitting) only allow half of the supplied nutrients to each biofilm during its growth phase. (B) Model prediction and (C) experimental validation of the average growth rate for a single biofilm (gray line) and for a biofilm pair (black line) at different glutamate concentrations. a.u., arbitrary units. (D) Biofilm growth rate is determined by the phase difference between biofilm pairs. Pairs of wild-type (solid line), ΔtrkA (dashed line), and ΔgltA (dotted line) biofilms all showed faster growth with antiphase oscillations (time-sharing) than with in-phase oscillations (resource-splitting). The color shading indicates glutamate concentration. Error bars represent SEM (n = 3 experiments).