Cooperation and conflict in microbial biofilms

Abstract

Biofilms, in which cells attach to surfaces and secrete slime (polymeric substances), are central to microbial life. Biofilms are often thought to require high levels of cooperation because extracellular polymeric substances are a shared resource produced by one cell that can be used by others. Here we examine this hypothesis by using a detailed individual-based simulation of a biofilm to investigate the outcome of evolutionary competitions between strains that differ in their level of polymer production. Our model includes a biochemical description of the carbon fluxes for growth and polymer production, and it explicitly calculates diffusion-reaction effects and the resulting solute gradients in the biofilm. An emergent property of these simple but realistic mechanistic assumptions is a strong evolutionary advantage to extracellular polymer production. Polymer secretion is altruistic to cells above a focal cell: it pushes later generations in their lineage up and out into better oxygen conditions, but it harms others; polymer production suffocates neighboring nonpolymer producers. This property, analogous to vertical growth in plants, suggests that polymer secretion provides a strong competitive advantage to cell lineages within mixed-genotype biofilms: global cooperation is not required. Our model fundamentally changes how biofilms are expected to respond to changing social conditions; the presence of multiple strains in a biofilm should promote rather than inhibit polymer secretion.

Fig. 1.

Simplified representation of the pathway for growth on glucose of an EPS-producing strain (EPS+) (a) and a corresponding nonproducer (EPS−) (b). Triple dots represent pathway intermediaries whose concentration is assumed to be stationary. In this pathway, glucose uptake is the rate-limiting step, and it has the same expression for both EPS+ and EPS− strains. EPS− has a higher intrinsic growth rate by directing its carbon flux to biomass, as opposed to dedicating part of the flux to EPS synthesis as in the case of EPS+.

Fig. 2.

Direct competition between an EPS+ and EPS− showing the importance of oxygen gradients in the outcome of the competition. (a–d) Simulation (a–c) and competition outcome (d) in the unrealistic case of absence of oxygen gradients. In this case, competition is decided purely by growth rate, and the fast-growing strain (EPS−) wins. (e–h) Simulation (e–g) and competition outcome (h) in the presence of oxygen gradients, showing the advantage to the EPS+ strain. Note, however, that the EPS+ strains only gain an advantage after a few days of growth so that for transiently formed biofilms, the EPS+ strategy will not be favored. (a–c and e–g) Oxygen concentration is shown in the background, where thick isoconcentration lines are shown for 1 mg/liter steps, and thin gray isoconcentration lines represent order of magnitude changes in the concentration (i.e., 0.1 mg/liter, 0.01 mg/liter, 0.001 mg/liter, etc.). These simulations where carried out at f = 0.55 for the EPS+ strain and ρ_X/ρ_EPS = 6. See also SI Movies 1–3.

Fig. 3.

Relative growth rate of an EPS+ in a biofilm with an equal starting frequency of an EPS−. Competition outcome depends on both the rate of EPS production of the producing strain and the density of EPS (ρ_X/ρ_EPS). Importantly, the EPS− often loses despite its higher intrinsic growth rate. Each box and whisker set results from 10 replicate simulations. Lines are a curve fitting using results from the 10 replicates. The dashed line represents the fitness of value 1, i.e., the borderline value above which the competition is won by EPS+.

Fig. 4.

Rare-mutant invasion analysis. (a) Invasion of a rare polymer-producing strain (EPS+) into a population of nonproducers (EPS−). (b) Invasion of a rare EPS− strain into a population of EPS+ strains. The x axis captures the number of strains randomly settling to form biofilms, where increased strains (and reduced within-group relatedness) cause a lower initial frequency of the rare mutant. For example, if 10 strains are present in all biofilms, the initial frequency of a rare mutant will be 0.1. All simulations were carried out at f = 0.5. Each box and whisker set results from 20 replicate simulations.

Fig. 5.

Polymer production by a founding cell is altruistic to the cells above but spiteful to adjacent cells (38, 39). Simulations were initiated with a single polymer producer (EPS+) surrounded by nonproducers (EPS−) (a) Plot of fitness of founding cells after 20 days of biofilm growth. The lineage of the focal cell (rightmost bar) benefits greatly from polymer production, whereas neighboring cells are suffocated and have reduced fitness. Fitness is calculated as the total number of cells that result from a focal cell in the mixed biofilm, and it is shown here as relative to a cell in a biofilm of pure nonpolymer producers (EPS−; dotted line). (b) Polymer producers are typically very successful in the mixed biofilms. (c) In a minority of cases, the polymer producers are smothered by nonproducers, which explains the high variance in the fitness of the focal polymer-producing cell (a). (d) Polymer production in biofilm has a strong analogy with growth in plants, where competition for light favors vertical growth.