Metabolic co-dependence gives rise to collective oscillations within biofilms

Abstract

Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigate such an internal conflict within a microbial (Bacillus subtilis) biofilm community: cells in the biofilm periphery not only protect interior cells from external attack but also starve them through nutrient consumption. We discover that this conflict between protection and starvation is resolved through emergence of long-range metabolic co-dependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth.

Extended Data Figure 1

Characterization of biofilm growth oscillations. a, (Top) Growth rate over time of an oscillating colony. (Bottom) The pressure that drives media flow in the microfluidic chamber is constant over time (see Methods: Microfluidics). b, (Top) Growth rate of an oscillating colony. (Bottom) Period of each oscillation cycle, measured peak to peak. The error bars (±20 min) are determined by the imaging frequency (1 frame/10 min). The period slightly increases over time (see also Extended Data Fig. 6f and Supplementary Information: Mathematical Model).

Extended Data Figure 2

Roles of carbon and nitrogen in biofilm growth oscillations. a, Effect of increasing carbon (glycerol) or nitrogen (glutamate) availability on the oscillations. While increasing glutamate by 5 times of the normal MSgg levels leads to quenching of the oscillation, increasing glycerol by 5 times does not. b, Colony growth of mutant strain with rocG deletion. B. subtilis NCIB 3610 has two glutamate dehydrogenases (GDH), rocG and gudB. While gudB is constitutively expressed, rocG expression is subject to carbon catabolite repression. The oscillatory growth of the rocG deletion strain indicates that carbon-source dependent regulation of rocG expression is not required for biofilm oscillations.

Extended Data Figure 3

Fourier transform of biofilm growth rates before and after addition of a, 1 mM glutamine, b, 1 mM ammonium, and c, 1 mM IPTG to induce Phyperspank-RocG. The error bars show standard deviations (n = 3 colonies for each condition). The arrows indicate the frequency of oscillations for each condition before perturbation (left) and the lack of oscillations after perturbation (right).

Extended Data Figure 4

Measurements of cell growth within oscillating biofilms. a, (Top) Visual representation of the method through which difference movies are generated (Methods: Data Analysis). Growth is represented by white pixels, and lack of growth is indicated by black pixels. (Middle) Film strip and (bottom) growth area over time of an oscillating colony. Dashed lines show the position of each image on the time trace. Scale bar represents 100 µm. b, (Top left) schematic of a biofilm. (Top right) high magnification phase contrast image of biofilm periphery focused at the bottom layer of cells. (Bottom panel) time traces depicting elongation rates of single cells in gray. Highlighted in red is the single cell time trace for the cell outlined in red in the top right panel. The periodic slowdown of the growth of individual peripheral cells is responsible for the observed periodic reduction in biofilm expansion.

Extended Data Figure 5

Effects of external ammonium on biofilm development. a, Addition of external ammonium (red shading, 1 mM) represses expression from the PnasA-YFP reporter (black), but does not affect expression from a constitutive reporter (Phyperspank-CFP + 1 mM IPTG, gray). b, Removal of external ammonium (red shading, 13 mM) causes halting of colony growth.

Extended Data Figure 6

Mathematical model of biofilm growth. a, The model describes the dynamics of two cell populations in a biofilm, interior and peripheral. As the biofilm grows, there is a constant distance between the interior population and the biofilm edge. b–e, Bifurcation diagrams showing systematic analysis on the effects of external glutamine, external glutamate, ammonium uptake, and GDH overexpression respectively. The red lines correspond to the extrema of oscillations in peripheral glutamate (stable limit cycle). The solid black line denotes stable fixed point. The dashed black line corresponds to an unstable fixed point. The vertical gray lines highlight the state of the system for each nutrient addition experiment shown in Fig. 3 of the main text. f, Model prediction of oscillation period as function of interior cell fraction in the whole biofilm. g–h, Sensitivity analysis of oscillation period and modulation depth to changes in model parameters. Modulation depth is defined as the amplitude of the oscillations divided by the mean value. Gray color denotes parameter regions where the system does not oscillate.

Extended Data Figure 7

Temporal profile of cell death within an oscillating biofilm. a, Colony growth rate. b, Average fluorescence intensity of a cell death marker (Sytox Green, 1 µM, Life Technologies) from the same colony shown in (a).

Extended Data Figure 8

Effect of external attack with hydrogen peroxide (H₂O₂, 0.15% v/v) or chloramphenicol (CM, 5 µg/ml). (Top) cell death shown by Sytox Green (1 µM). (Middle and bottom) colony growth shown by image differencing (see Extended Data Fig. 4a and Methods: Data Analysis). Scale bar represents 100 µm. The white dashed lines indicate colony edge.

Extended Data Figure 9

Effect of GDH induction on cell growth. Wild type and Phyperspank-RocG (uninduced or induced with 10 mM IPTG) strains were grown in liquid culture (MSgg medium, 30°C). Cell generation times were measured using OD₆₀₀. Error bars show standard deviations (n = 3 replicates).

Extended Data Figure 10

Growth rate oscillations persist in various mutant strains. a, opp operon deletion (deficient in quorum sensing). b, comX deletion (deficient in quorum sensing). c, tapA operon deletion (extracellular matrix component deletion). d, tapA operon overexpression (Phyperspank-tapA operon, 1mM IPTG). e, hag deletion (deficient in swimming and swarming). These results show that the corresponding genes and processes are not required for biofilm oscillations.

Figure 1

Biofilms grown in microfluidic devices show oscillations in colony expansion. a, Biofilms must reconcile opposing demands for protection from external challenges (gradient indicated in purple) and access to nutrients (gradient indicated in gray). b, Schematic of the microfluidic device used throughout this study. Direction of media flow is indicated by the blue arrows. c, Phase contrast image of a biofilm growing in the microfluidic device. The yellow arrow indicates the region of interest in panel (d). d, Filmstrip of a radius of the biofilm over time shows a pause in colony expansion. This film strip represents one cycle of biofilm oscillations, indicated by the shaded region in panel e. Scale indicates 5 µm. e, Growth rate over time shows persistent oscillations in colony expansion. f, Histogram of the average period of oscillations for each colony (n = 63 colonies, mean = 2.5 hours, s.d. = 0.8 hours). The cell replication time is approximately 3.4 hours under these conditions (Methods: Data Analysis). g. Growth rate as a function of colony diameter (which increases in time) shows that early colony growth does not exhibit oscillations. The orange line indicates the diameter (~600 µm) at which this colony initiates oscillations. h, Histogram of the diameter at which a colony begins to oscillate (n = 53 colonies, mean = 576 µm, s.d. = 85 µm).

Figure 2

Biofilm growth depends specifically on extracellular ammonium availability. a, Colony growth in MSgg medium depends on the production of glutamine from externally supplied glutamate and self-produced or scavenged ammonium. Glutamine limitation was monitored using YFP expressed from the nasA promoter, which is activated upon glutamine limitation. b, Addition of 1 mM glutamine (blue shading) represses expression from the PnasA-YFP reporter (black), but does not affect expression from a constitutive reporter (Phyperspank-CFP + 1 mM IPTG, gray). c, Growth area (see Methods: Data Analysis) before and after addition of 1 mM glutamine to an oscillating colony. d, Of the two nutrients required for glutamine production, externally supplied glutamate (green) is most abundant in the biofilm periphery, while biofilm-produced ammonium (red) is most abundant in the biofilm interior. e, Maximum intensity projection over one period of a colony oscillation, made from a difference movie (Methods: Data Analysis), which shows regions of growth (white) and no growth (black). Scale bar represents 100 µm. f, Growth area of an oscillating colony before and after addition of 30 mM glutamate (green shading). g, Growth area of an oscillating colony before and after addition of 1 mM ammonium (red shading).

Figure 3

Mathematical modeling of a spatial metabolic feedback loop gives rise to oscillations consistent with experimental data. a, The production of ammonium in the interior is limited by and at the same time triggers the consumption of glutamate in the periphery (green and red arrows, respectively), producing a delayed negative feedback loop. b, The excess glutamate not consumed by the biofilm periphery diffuses to the interior, where it can be converted into ammonium (green arrows). The ammonium in turn enhances growth in the periphery (red arrow) and consequently reduces the supply of glutamate to the interior. Model predictions are shown in (c–h): c, Biofilm growth over time. d, Glutamate concentration over time. e, Ammonium concentration over time. f, Colony growth before and after glutamine addition (indicated by blue shading). g, Colony growth before and after addition of glutamate (green shading). (h) h, Colony growth before and after addition of ammonium (red shading).

Figure 4

Metabolic codependence between interior and peripheral cells gives rise to oscillations that make the colony more resilient to external attack. a, Visual representation of the predicted outcome of an external attack on biofilm growth. b, Phase contrast merged with cell death marker (cyan, 1 µM Sytox Green) images of a wild type biofilm region shows cell death with and without challenge by 2% w/w H₂O₂. Scale bar represents 50 µm. c, In the same biofilm, difference images (white regions indicate cell growth) show wild type growth with and without challenge by H₂O₂. d, Overexpression of glutamate dehydrogenase (GDH, pink) promotes more production of ammonium from glutamate. e, Experimental (top) and modeling results (bottom) of GDH overexpression (induced with 1 mM IPTG, indicated by pink shading). f, Phase contrast merged with cell death marker (cyan, 1 µM Sytox Green) images of a colony overexpressing GDH with and without challenge by H₂O₂. g, In the same biofilm, difference images show cell growth during GDH overexpression alone, and with challenge by H₂O₂. h, Quantification of total biofilm growth rate in wild type (upper, n = 4 colonies) and GDH overexpression (lower, n = 3 colonies) strains upon challenge with H₂O₂. Error bars represent standard deviations. Modeling data are shown as an inset for each strain. i, Codependence between interior and peripheral cells exhibited in a wild type strain results in a growth strategy that sustains the viability of interior cells, while independence enforced by a GDH overexpression strain results in starvation of interior cells and reduced resilience to external attack.