Fig. 3: A putative AlpA binding element is located between the −35 and −10 elements of target promoters.
From: Control of a programmed cell death pathway in Pseudomonas aeruginosa by an antiterminator
a Sequence of alpB and PA0807 promoters and point mutations made in ABE6 and ABE7 promoter mutants. Mutations colored in green, −35 and −10 elements highlighted in gray, TSS in blue. Putative AlpA binding motif is GGGACGN(5)GGTA. b Effect of ABE6 mutation on AlpA-dependent control of a PalpB tB lacZ transcriptional reporter in PAO1. c Effect of ABE7 mutation on AlpA-dependent control of a PPA0807 lacZ transcriptional reporter in PAO1. b, c Cells of the indicated reporters containing plasmids pAlpA or pPSV38 (the empty vector control, indicated pEV) were assayed for β-galactosidase activity, which is shown in Miller Units. 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. PalpB with pEV vs. pAlpA: p = 1.1 × 10−8. PalpBABE6 with pEV vs. pAlpA: p = 8.5 × 10−12. PPA0807 with pEV vs. pAlpA: p = 5 × 10−8. PPA0807ABE7 with pEV vs. pAlpA: p = 6.6 × 10−6; p ≤0.0001 = ****. d, e Effect of ABE6 and ABE7 mutations on association of AlpA with the respective alpB and PA0807 promoter regions as determined by ChIP-qPCR. 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. PAO1 (WT PalpB) vs. PalpBABE6: p = 0.01. PAO1 (WT PPA0807) vs. PPA0807ABE7: p = 0.0059. p-Values indicated by the following symbols: ≤0.01 = **. f Ectopic synthesis of the AlpR-CTD results in cell death or inhibition of growth in WT PAO1 and PAO1 harboring the ABE7 mutation in the AlpA binding site of the endogenous PA0807 promoter (PPA0807ABE7) but not in PAO1 with a deletion of alpBCDE or in PAO1 harboring the ABE6 mutation in the endogenous alpB promoter (PalpBABE6). Plasmid pAlpR-CTD directs the synthesis of the AlpR-CTD in an IPTG-inducible manner. Indicated PAO1 strains were serially diluted tenfold and incubated on media lacking or containing IPTG. g Time-lapse microscopy images at the indicated time points of PAO1 mCherry with a WT alpB promoter (synthesizing mCherry and shown in red) and PAO1 GFP PalpBABE6 (not synthesizing mCherry, shown in phase contrast) ectopically synthesizing the AlpR-CTD. Scale bar is 10 μm. Experiment was performed independently twice with similar results. Source data are provided as a Source Data file.
