a, Tis1-depleted cells exhibited a reduction in viability over time. CFU plating of P. aeruginosa PA14 ΔretSΔsspB Tis1-D4 pPSV39::sspB cells at the indicated times after 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 times. The parental PA14 strain genotype is ΔrsmAΔrsmF. 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 harbouring the Tas1 tox expression plasmid in LB medium with or without CCCP. Curves for n = 3 cultures are overlaid for each 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 before induction or at the indicated times after induction. e, Alkaline pH does not affect the ability of CCCP to reduce Tas1 tox -dependent toxicity, indicating that the toxicity of Tas1 tox is likely to arise from the generation of excessive membrane electrostatic potential. Cultures were untreated or conditioned to pH 8.0 using 25 mM Tris-HCl buffer immediately before 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. Mean ± s.d. for enzymatic activity from n = 4 technical replicates; two-tailed, unpaired t-test. g, Anion-exchange chromatography traces of metabolites extracted from growth competition experiments between the indicated strains conducted on solid medium for 4 h. The parental strain is ΔrsmAΔrsmF. Traces are representative of three independent experiments. a, b, d, e, Plates are representative of three independent experiments.