Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Abstract

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.

Figure 1

B. thailandensis–C. violaceum competition. Initial cell densities were 2–4 × 10⁷ B. thailandensis (Bt) cells per ml and 2–4 × 10⁶ C. violaceum (Cv) cells per ml. The initial and final cell densities of Bt and Cv were determined for each independent experiment by selective plating and colony counts. Each data point represents the log-transformed average of the ratios of the two species from duplicate measurements of an independent co-culture experiment. The lines represent the mean of all of the experiments in each set.

Figure 2

Competition in co-cultures of wild-type C. violaceum (Cv) and wild-type or mutant B. thailandensis (Bt) strains. The dashed line indicates the starting 10:1 ratio of Bt to Cv. The ratio of Bt to Cv after 24 h was determined by selective plating and colony counts. The co-culture results with wild-type Bt are also shown in Figure 1 and the final average CFU of each species is also partially represented in Table 3. Bt AHLs were extracted from culture fluid of a Bt bactobolin mutant (see Materials and methods) and added to culture medium. The solid lines represent means for each group. The vertical bars show the standard error of the mean for each group.

Figure 3

Co-cultures of the C. violaceum (Cv) wild-type Cv017 or the AHL mutant Cv026 and the B. thailandensis (Bt) competition-impaired AHL, bactobolin double mutant JBT125. The dashed line shows the initial ratio of Bt to Cv. After 24 h, the ratio of Bt to Cv was determined by colony counts on selective agar. Co-cultures were grown in 20 ml medium. C6-HSL was added before inoculation where indicated (250 nℳ final concentration). The solid lines represent the means of each group.

Figure 4

C. violaceum (Cv) quorum sensing is activated by B. thailandensis (Bt) AHLs. Quorum-sensing activation is indicated by the Cv quorum-sensing-dependent purple pigment, violacein, in stationary-phase cultures. (a) Cv wild-type (Cv017) and the AHL mutant (Cv026) with or without added Bt AHLs. (b) Co-cultures of the Cv AHL mutant and Bt strains as indicated (AHL mutant JBT112; AHL, bactobolin double mutant JBT125). AHLs were extracted from stationary-phase cultures of Bt BD20, a bactobolin mutant.

Figure 5

Eavesdropping promotes competitiveness of Cv in co-cultures with a B. Bt bactobolin mutant. After 24 h of co-culture, the ratio of Bt to Cv was determined. Co-cultures of the Cv AHL mutant (Cv026), or the Cv AHL synthase, receptor double mutant (Cv026R) and the Bt strains as indicated and described in Figure 4 legend. Co-cultures were grown in 20 ml volumes. The dashed line indicates the initial ratio of Bt to Cv. The solid lines represent the means for each group. AHLs were obtained as described for Figure 4.

Figure 6

In silico modeling. Our model accounts for two species with quorum-sensing-controlled antibiotics, similar to our experimental model of Bt and Cv. As in our experimental model, our in silico model accounts for two species (B and C) that produce antibiotics in a density-dependent manner. In our model, species C can eavesdrop on species B (see Supplementary Text). We show relative fitness of each species as a function of the initial ratio (C/B) and the eavesdropping sensitivity (ɛ) of C. The fitness of C relative to B was measured using the log relative fitness measure given in Wu et al. (2006) and is indicated by the color spectrum on the far right. (a) The inducer concentration required for production of antibiotic (activation threshold, K_B and K_C) is relatively high for both species (0.01, see text). (b) The activation threshold is lower (0.003898) and corresponds to an optimal threshold for each species that gives it an advantage over the other species regardless of the other species' threshold. (c) Both species have an activation threshold lower than the optimal threshold (0.003). (d) The same parameters were used as in (c); however, the antibiotic toxicity is raised 10-fold. This changes the optimal activation threshold to 0.001113, which is below the activation threshold value used (0.003) (see Supplementary Figure S1).