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.