High local substrate availability stabilizes a cooperative trait

Abstract

Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.

Figure 1

Localized peptide concentrations measured by intracellular luciferase-based peptide sensing. Mixed batch cultures of prt⁺ and prt⁻ strains were grown in reconstituted skimmed milk. The fraction of the prt⁺ strains in the co-culture were 90% (a, b), 50% (c, d) and 10% (e, f). Intracellular peptide concentrations were measured via the dppA-controlled luminescence signal and are given in arbitrary units (y axis, top panel). The dppA reporter construct resides either in the prt⁺ (dashed line) or the prt⁻ (solid line) host strain. The presented data is corrected for the relative abundance of the strain carrying the luciferase reporter. The slopes of the luminescence traces are given on the y axis of the lower panels. Each curve represents the average of four biological replicates. The results show that at a high relative abundance of the prt⁺ strain, the dppA expression levels are similar, indicating little or no difference in peptide availability for the two strains (a–d). At low frequencies of the prt⁺ strain, the intracellular amino-acid levels are higher in the prt⁺ strains (e, f), which is detected by the downregulation of dppA expression in that strain. For the lower panels, the standard error is shaded in gray. The dip in luminescence activity between 400 and 500 min on the x axis is an intrinsic property of the luminescence reporter, which is linked to changes in metabolic activity when cells go into stationary phase (Bachmann et al., 2007).

Figure 2

(a) Modeling population dynamics of prt⁺ and prt⁻ mixed strain cultures. The heat map displays the fractional gain of the prt⁺ strain after one culturing step. The dependency of this fractional gain on the initial fraction of the prt⁺ cells in the culture (x axis) and the inoculation density in colony forming units (CFU) (y axis) is shown. With increasing inoculation densities and/or an increasing fraction of the prt⁺ strains in the culture, the overall advantage of the prt⁺ strain vanishes. Model and parameters are given in the supplementary information. (b) Relative fitness (y axis) of prt⁺ strains if propagated at different cell densities. Equal amounts of prt⁺ and prt⁻ strains were inoculated in milk, and propagated for about 100 generations. The inoculation densities at each propagation event are indicated (x axis). For each condition, three biological replicates were propagated (o). Linear regression shows a highly significant correlation between the fitness of prt⁺ strains and the inoculation density (R²=0.91, P<0.0001). The prt⁺ strain could be stabilized in the culture when propagated at low cell densities (W∼1) and their abundance in the culture decreased when propagated at high cell densities (W<1).