A Metabolic Trade-Off Modulates Policing of Social Cheaters in Populations of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of public goods such as the secreted protease elastase. P. aeruginosa requires the LasI-LasR QS circuit to induce elastase and enable growth on casein as the sole carbon and energy source. The LasI-LasR system also induces a second QS circuit, the RhlI-RhlR system. During growth on casein, LasR-mutant social cheaters emerge, and this can lead to a population collapse. In a minimal medium containing ammonium sulfate as a nitrogen source, populations do not collapse, and cheaters and cooperators reach a stable equilibrium; however, without ammonium sulfate, cheaters overtake the cooperators and populations collapse. We show that ammonium sulfate enhances the activity of the RhlI-RhlR system in casein medium and this leads to increased production of cyanide, which serves to control levels of cheaters. This enhancement of cyanide production occurs because of a trade-off in the metabolism of glycine: exogenous ammonium ion inhibits the transformation of glycine to 5,10-methylenetetrahydrofolate through a reduction in the expression of the glycine cleavage genes gcvP1 and gcvP2, thereby increasing the availability of glycine as a substrate for RhlR-regulated hydrogen cyanide synthesis. Thus, environmental ammonia enhances cyanide production and stabilizes QS in populations of P. aeruginosa.

Keywords: LasR; RhlR; hydrogen cyanide; quorum sensing; sociomicrobiology.

FIGURE 1

Emergence of LasR-mutant cheaters and their fitness in PM-casein with or without ammonium sulfate added. (A) Percent of protease-negative cheaters in PM-casein media over time. In four of five individual experiments without added ammonium sulfate, cheaters emerged and increased (hollow symbols). In PM-casein with ammonium sulfate (black symbols), cheaters reached a frequency of 27 ± 7%. In one experiment with and one experiment without ammonium sulfate, cheaters did not emerge. (B) Cell densities in casein experiments. In the cases where the culture could not be propagated, the final cell densities fell by 90–99% (hollow symbols). Experiments in PM-casein with ammonium sulfate (black symbols) are shown for comparison. (C) LasR-mutant frequency after 7 days of passage. LasR-mutant cheater frequency in PM-casein without ammonium sulfate was rapid in all five replicates from an initial frequency of 1% (hollow symbols). An equilibrium was always established (black triangles) in media with ammonium sulfate added. When LasR mutants were not present at the beginning of the experiment, they did not emerge in 7 days PM-casein with ammonium sulfate (black circles). (D) Outcomes of w.t. – LasR-mutant competitions with increasing ammonium sulfate concentrations. w.t. PAO1 and LasR-mutant cheaters were mixed at a 1:1 ratio and the frequency of each was enumerated after 24 h. Experiments were performed in triplicate. For the data in panel D, a linear regression analysis was performed.

FIGURE 2

LasR and RhlR activities in w.t. P. aeruginosa. (A) GFP transcription in w.t. containing the lasI–gfp reporter plasmid pBS351. GFP is a proxy for LasR activity. Added ammonium sulfate does not alter the LasR activity of cooperators in w.t or a RhlR mutant, either in the presence or absence of cheaters. (B) GFP transcription in w.t. using the rhlA–gfp reporter plasmid pJF01. Addition of ammonium sulfate significantly increased the RhlR activity of cooperators in presence of cheaters. A RhlR mutant is shown as a reference. (C) Transcription of lasR and rhlR measured by qRT-PCR. Added ammonium sulfate significantly increased the transcript levels of rhlR but not lasR in the presence of cheaters. (D) Added ammonium sulfate significantly increased the expression of rhlR in the presence of LasR-mutant cheaters. The w.t. and LasR mutants were separated by a dialysis membrane. “Mono”: monoculture of w.t. or the RhlR mutant. “Co”: co-culture with 9:1 mixture of w.t and LasR mutants or a mixture of RhlR mutants and LasR mutants. We used proC as a reference (housekeeping) gene. White bars indicate PM-casein without ammonium sulfate and black bars indicate PM-casein with ammonium sulfate. Experiments were performed in triplicate. Data reported as the mean ± SD. ^∗ indicates p < 0.05 by paired Student’s t-tests.

FIGURE 3

Ammonia promotes cyanide and ROS production by cooperators. (A) hcnC transcription in w.t. increases in presence of LasR-mutant cheaters in PM-casein with ammonium. hcnC expression from either a RhlR or HcnC deletion mutant in co-culture with the LasR mutant is shown as controls. proC was used as reference gene. In panels A–C, white bars indicate media without ammonium sulfate and black bars indicate that ammonium sulfate was added. (B) Cyanide production by cooperators in PM-casein with or without ammonium sulfate. (C) ROS production from LasR mutants in LB-MOPS, combined with supernatants of strains grown in PM-casein media. “-” indicates no cells. (D) ROS production by the w.t. increases as the concentration of ammonium sulfate is increased. (E) Growth inhibition of the LasR mutant by supernatant from w.t.:LasR-mutant co-culture from PM-casein with ammonia media (inverted triangle). The supernatant from a w.t.:LasR-mutant co-culture in PM-casein media (triangle) did not inhibit the growth of LasR mutants; neither did supernatant from a HcnC-mutant: LasR-mutant co-culture (square). The arrow indicates the time of supernatant addition. Experiments were performed in triplicate. ^∗ indicates p < 0.05 by paired Student’s t-tests (panels A–C and E). A linear regression analysis was performed for the data presented in panel D.

FIGURE 4

Exogenous ammonia inhibits the transformation from glycine to 5,10-methylene-THF. (A) Expression of gcvP1 and gcvP2 by the w.t. in a co-culture with 10% LasR mutants. Hollow bars indicate PM-casein without ammonium sulfate and black bars indicate PM-casein with ammonium sulfate. proC was used as a housekeeping gene. (B) PAO1 generates ammonia faster in the absence of ammonium sulfate (hollow circles) than in its presence (black circles). (C) Model of the flux between exogenous ammonia and cyanide production in P. aeruginosa. In panel C, red stars indicate ROS. The red NH₄⁺ indicates exogenous ammonia in media, and black text indicates released ammonia by metabolism. Experiments were performed in triplicate. ^∗ indicates p < 0.05 by paired Student’s t-test (panel A) or one-way ANOVA (panel B).