Extended Data Figure 9 |. The pmf uncoupling ionophore CCCP but not the ppGpp-hydrolase domain of SpoT reduces the toxicity of Tas1_tox.

a) Tis1-depleted cells exhibited a reduction in viability over time. CFU plating of P. aeruginosa PA14 ΔretS ΔsspB Tis1-D4 pPSV39::sspB cells at the indicated time points post induction of SspB expression. b) Tas1 reduces the viability of susceptible recipient cells during interbacterial competition. CFU plating of the indicated P. aeruginosa PA14 recipient strains after co-culture with a parental donor strain at the indicated time points. The parental PA14 strain genotype is ΔrsmA ΔrsmF. c) Steady-state growth of E. coli is not substantially affected by the presence of carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Growth curves of E. coli cells harboring the Tas1_tox expression plasmid in LB medium with or without (CCCP). Results from three independent cultures were overlaid for each medium condition. d) Tas1_tox toxicity is reduced in the presence of CCCP. Viability of E. coli cells following Tas1_tox expression in the presence or absence of CCCP. Cells were plated either pre-induction or at the indicated times post-induction. e) Alkaline pH does not affect the ability of CCCP to reduce Tas1_tox-dependent toxicity indicating that the toxicity of Tas1_tox likely arises from the generation of excessive membrane electrostatic potential. Cultures were untreated or conditioned to pH 8.0 using 25 mM Tris-HCl buffer immediately prior to induction. f) Activity of the ppGpp-hydrolase domain of SpoT against either ppGpp or ppApp. Initial velocities were normalized to hydrolase activity in the absence of either nucleotide. Data are mean ± SD for enzymatic activity from four technical replicates. P value from two-tailed, unpaired t-test is shown. g) Anion-exchange chromatography traces of metabolites extracted from growth competition experiments between the indicated strains conducted on solid media for 4 hours. The parental strain is ΔrsmA ΔrsmF. Traces are representative of three biological replicates. a-b, d-e) Representative plates of three biological replicates are shown.