Fig. 6: ppGpp modulates AlpA activity in P. aeruginosa and in E. coli.
From: Control of a programmed cell death pathway in Pseudomonas aeruginosa by an antiterminator
a qRT-PCR was used to measure transcript abundance of genes in the two AlpA-regulated operons in strains that have alpB deleted or alpB and relA deleted. ∆alpB was used as the background strain to prevent lysis of cells ectopically synthesizing the AlpR-CTD. In ΔrelA mutant cells that contain the alpR-CTD expression vector pAlpR-CTD, there is a threefold decrease in transcript abundance of PA0807 (gray) and twofold decrease in transcript abundance of alpD (gold) compared to WT (i.e. relA+) cells. Values and error bars reflect mean ± SD of n = 3 biological replicates. Two-tailed, unpaired, unequal variance t-tests were used to calculate p-values between indicated samples. ∆alpB ∆relA (pAlpR-CTD) vs. ∆alpB (pAlpR-CTD) for the PA0807 transcript: p = 0.0078. ∆alpB ∆relA (pAlpR-CTD) vs. ∆alpB (pAlpR-CTD) for the alpD transcript: p = 0.019. p-Values indicated by the following symbols: >0.05 = ns, ≤0.05 = *, ≤0.01 = **. b Image shows tenfold serial spot dilutions of cells of indicated strains of P. aeruginosa (PAO1 is WT) ectopically expressing the alpR-CTD under the control of an IPTG-inducible promoter from plasmid pAlpR-CTD. Ectopic expression of alpR-CTD is lethal due to activation of alp encoded PCD pathway. Deletion of alpBCDE suppresses lethality of ectopic alpR-CTD expression. PAO1 cells with relA and spoT deleted survive ectopic expression of alpR-CTD better that WT cells but not as well as cells of the ∆alpBCDE mutant strain. c β-galactosidase activity (in Miller Units) of the indicated reporter constructs in E. coli. Promoters were either lacUV5 or alpB and strain backgrounds were either WT and could produce ppGpp (indicated WT) or were relA spoT mutants that cannot make ppGpp (indicated relA spoT). Cells contained plasmid pAlpA (gold) or plasmid pEV (the empty vector; gray). Values and error bars reflect mean ± SD of n = 3 biological replicates in technical duplicate. Two-tailed, unpaired, unequal variance t-tests were used to calculate p-values between indicated samples. WT PalpB (pAlpA) vs. relA spoT PalpB (pAlpA): p = 7.6 × 10−8. p-Values indicated by the following symbols: >0.05 = ns, ≤0.0001 = ****. Source data are provided as a Source Data file.
