Costs and benefits of provocation in bacterial warfare

Abstract

Competition in animals involves a wide variety of aggressive behaviors. One of the most sophisticated strategies for a focal actor is to provoke a competitor into uncontrolled aggression toward other competitors. Like animals, bacteria rely on a broad spectrum of molecular weapons, some of which provoke potential rivals by triggering retaliation. While bacterial provocation is well documented, its potential adaptive value has received little attention. Here, we examine the costs and benefits of provocation using mathematical modeling and experiments with Escherichia coli strains encoding colicin toxins. We show that provocation is typically costly in one-to-one encounters because a provoking strain receives a strong reciprocal attack compared with nonprovoking strains. By contrast, provocation can be strongly beneficial in communities including more than two toxin-producing strains, especially when the provoker is shielded from, or resistant to, its opponents' toxins. In these scenarios, we demonstrate that the benefit of provocation derives from a "divide-and-conquer" effect by which aggression-provoking toxin producers force their competitors into increased reciprocal aggression, leading to their cross-elimination. Furthermore, we show that this effect can be mimicked by using antibiotics that promote warfare among strains in a bacterial community, highlighting the potential of provocation as an antimicrobial approach.

Keywords: bacterial communities; colicin; competition; provocation; social evolution.

Fig. 1.

Aggression-provoking strains are at a disadvantage in one-to-one competitions against other toxin-producing competitors. (A) E. coli strains increase their toxin production when exposed to colicins with DNase, but not other, activities. Cultures of a colicin-E1–producing strain were exposed to spent media from four strains for 3 h. Extracts of cells were then tested for toxicity using a growth inhibition assay. (B) Strains producing aggression-provoking toxins are at a disadvantage in one-to-one competitions with other toxin-secreting strains. The graphs show the dynamics of the modeled population in pairwise encounters between strains of three types: sensitive nonproducer (cyan), nonprovoking toxin producer (black or dark gray), and aggression-provoking toxin producer (red). Against a nonprovoking toxin producer, the aggression-provoking strain (Right) decreases faster than the sensitive strain (Left). In all ODE models in this study, the maximal growth, lysis, and toxin production rates, as well as the general toxin characteristics, are identical across strains. (C and D) E. coli strains producing colicins with DNase activity are at a disadvantage when competing one-to-one against other colicin-producing strains. (C) Pairs of colicinogenic strains were spotted onto LB agar, each at a different dilution (1 vs. 10⁻³, respectively), and grown overnight. (D) The area occupied by the spot of the more dilute, fluorescent strain was recorded. Large and small letters on the graph correspond to the concentrated and dilute spot, respectively. For strain annotation, see Table 1; in all panels, “B” stands for BZB1011, a sensitive noncolicinogenic strain. Statistical tests can be found in SI Appendix. (Scale bars: 2,000 μm.)

Fig. 2.

Aggression-provoking strains benefit from provocation, provided they are shielded from or resistant to their competitors’ toxins. (A) We modeled competitions of three toxin-producing strains, including a focal strain segregated from its competitors or resistant to their toxins. Segregation means that the probability for the focal strain to reach or be reached by its competitors is lower by a factor u (segregation factor) compared with the probability of its two competitors to reach each other. The graph shows the ratio between the frequency of a focal provoker relative to a focal nonprovoker against the same competitors as a function of time for different values of u. For u > 2, an aggression-provoking strain benefits compared with a nonprovoker (gray lines). A resistant provoker has an even stronger advantage (red line). We note that the differences between provoker and nonprovoker appear for a limited time frame, after the toxins reach relevant concentrations and before the community stops growing because of nutrient exhaustion (SI Appendix, Fig. S12). (B and C) When segregated and opposed to mixed colonies of two colicin-producing strains, an E. coli strain producing a DNase colicin (E2) has a strong advantage compared with a strain producing a non-DNase colicin (A). (B) An undiluted focal strain was spotted onto LB agar next to a mixed spot of two other colicin producers, diluted 1,000-fold, and grown overnight. (C) The area occupied by the mixed spot was measured. For each focal strain (B, A, or E2), in the absence of interaction, the area occupied by the mixed colicin producers (E1E4) is expected to be no lower than the lowest of the values for single competitors (E1B or E4B). The disproportionate decrease in the case of E2 is evidence for provocation. (D and E) In communities of three toxin producers, a colicin-resistant E. coli strain producing a DNase colicin (E2) has an overwhelming advantage compared with a resistant strain producing a non-DNase colicin (A). (D) A mixture of three strains, a focal resistant strain labeled with red fluorescent protein (RFP) and each of its competitors alternatively labeled with GFP or unlabeled, was spotted onto LB agar and grown overnight. (E) The fluorescence intensity of each strain was recorded. The graph shows the ratios between the focal strain (B^R, A^R, and E2^R) and both of its competitors (E1 and E4), normalized to the focal nonproducer (B^R). For strain annotation, see Table 1; in all panels “B” stands for BZB1011, a sensitive noncolicinogenic strain. Statistical tests can be found in the SI Appendix. (Scale bars: 2,000 μm.)

Fig. 3.

Increase in aggression has a strong impact on the productivity and composition of bacterial communities. (A–C) The productivity of three-member communities is hampered when an aggression-provoking strain is present. (A) We modeled the competition of three-strain communities and report the dynamics of each strain (nonproducer in cyan, nonprovoking producers in black and dark or light gray, aggression-provoking producer in red) and of the whole population (thin gray line). While an aggression-provoking strain is frequent in the community (Right), the overall community productivity is heavily impacted (time points 1–15). (B) Equal amounts of three strains, each in turn labeled with GFP, were mixed and spotted onto LB agar. (C) After overnight incubation, the fluorescence intensity for each strain was measured, and intensities were summed up for each community (strains B, A, or E2 in white; first and second competitors in dark and light gray, respectively). (D and E) DNA-damaging chemicals have a similar effect on bacterial communities as aggression-provoking toxins. (D) Equal amounts of colicin-E1– and colicin-E4–producing strains (GFP- and RFP-labeled, respectively) were mixed and spotted, together with controls, onto LB agar with or without added mitomycin C at a final concentration of 0.01 μg ml⁻¹. After overnight incubation, the intensity of the fluorescence was measured. (E) The summed RFP and GFP fluorescence values are plotted. For strain annotation see Table 1; in all panels “B” stands for BZB1011, a sensitive non colicinogenic strain. Statistical tests can be found in the SI Appendix. (Scale bars: 2000 μm.)