Cross-species activation of hydrogen cyanide production by a promiscuous quorum-sensing receptor promotes Chromobacterium subtsugae competition in a dual-species model

Abstract

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.

Keywords: Chromobacterium; antibiotic; coculture; competition; cyanide; quorum sensing.

Fig. 1.

Burkholderia thailandensis–Chromobacterium subtsugae coculture model. The B. thailandensis quorum-sensing system BtaR2-I2 produces and responds to 3OHC8-HSL and 3OHC10-HSL, and activates production of bactobolin antibiotic. B. thailandensis also has two other quorum-sensing systems, BtaR1-I1 and BtaR3-I3, which produce C8-HSL and 3OHC8-HSL, respectively. BtaR2-I2 is important for B. thailandensis to compete with C. subtsugae due to activation of bactobolin production. The C. subtsugae quorum-sensing system CviR-I produces and responds to C6-HSL. CviR-I is important for C. subtsugae to compete with B. thailandensis, through an unknown mechanism.

Fig. 2.

Contribution of B. thailandensis AHLs to C. subtsugae competitiveness. Cocultures were with C. subtsugae ΔcviI and B. thailandensis ΔbtaI1,ΔbtaI2,ΔbtaI3,ΔbtaK (JBT125). After 24 h of coculture growth, the ratio of C. subtsugae to B. thailandensis was determined by selective plating and colony counts. The dashed line indicates the initial ratio of C. subtsugae to B. thailandensis . The solid line represents the means and the vertical bars shows the standard error of the mean for each group. Synthetic AHLs were added at the beginning of the coculture to a final concentration of 1 µM. Statistical comparisons of each condition with no signal coculture were by one-way ANOVA; ***, P<0.001; **, P<0.005; ns, not significant.

Fig. 3.

Cyanide measurements in C. subtsugae culture fluid. Cyanide (CN⁻) was measured in culture fluid of C. subtsugae . (a) Wild-type C. subtsugae (CV017) was grown to different population densities as indicated by the optical density at 600 nm (OD₆₀₀) prior to measuring CN⁻. Data are shown as growth-adjusted CN⁻. The total CN⁻ concentration at an OD₆₀₀ of ~8 corresponds to 3.3±1.1 mM. (b) The indicated C. subtsugae strain was grown for 12 h, which corresponds with an OD₆₀₀ of ~8, prior to measuring population density and CN⁻. Data are shown as the growth-adjusted CN⁻ as a percentage of that of wild-type. In all cases, data represent the average of three biological replicates and the vertical bars represents the standard error of the mean.

Fig. 4.

Cyanide toxicity and role in cocultures. (a) Survival of B. thailandensis BD20 grown in the presence of different concentrations of cyanide for 8 h from an initial OD₆₀₀ of 0.005. The population density was measured and B. thailandensis (Bt) survival was determined as a percentage of untreated culture grown under identical conditions. (b) Cocultures were with B. thailandensis ΔbtaK (BD20) and the C. subtsugae strain indicated. After 24 h of coculture growth, the ratio of C. subtsugae to B. thailandensis was determined by selective plating and colony counts. The initial ratio of C. subtsugae to B. thailandensis was 10⁻¹. The solid line represents the means and the vertical bars shows the standard error for each group. Statistical comparisons of each condition compared with no signal were by one-way ANOVA; **, P<0.005; * = P<0.05 and ns, not significant.

Fig. 5.

Sensitivity of the hcnA promoter to native and non-native AHLs. (a) C. subtsugae ΔcviI or ΔcviR mutant harbouring the hcnA-lacZ reporter was treated with 5 µM of AHL (C6-HSL, C8-HSL, 3OHC8-HSL or 3OHC10-HSL). (b) C. subtsugae ΔcviI harbouring the hcnA-lacZ reporter was treated with AHLs at the indicated concentrations. Results show the average of four independent experiments from two separate days, and the vertical bars represent the standard error.

Fig. 6.

Hydrogen cyanide biosynthesis genes (hcnABC) in C. subtsugae and P. aeruginosa . Red arrows and lettering indicate the transcription start site. The arrow is dashed for C. subtsugae , as it was predicted to be between nucleotides −79 to −81 (see Methods). Green boxes and lettering indicate the binding site for the anaerobic regulator (Anr) and the blue box and lettering indicate the lux box of P. aeruginosa, and the canonical lux box. Bolding in the P. aeruginosa lux box sequence indicates nucleotides that are conserved with the canonical lux box. No lux box or Fnr/Anr-like sequences were identified in the C. subtsugae sequence (see the Discussion).