DksA and ppGpp Regulate the σ^S Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP

DksA stimulates expression of IraP in vivo. Shown is β-Gal activity during exponential growth at 32°C in LB with 0.1 mM IPTG. (A) P_iraP-lacZ transcriptional fusion with pControl or pDksA_N88I in WT cells. (B) P_katE-lacZ transcriptional fusion in a ΔiraP mutant with pControl and DksA_N88I. The β-Gal data are representative of at least three independent experiments. The absolute activities in panels A and B should not be compared directly, since the fusions are to different promoters transcribed by different holoenzymes.

The effect of DksA_N88I on σ^S levels is dependent on dsrA. Strains were grown to an OD₆₀₀ of 0.6 in LB at 32°C with 0.1 mM IPTG. Shown is Western blot analysis of σ^S protein levels. (A) Protein extracts were made from the indicated strains containing pControl or pDksA_N88I, and σ^S was normalized to the WT with pControl. A representative Western blot is shown below the graph. (B) Proteins were extracted from WT or ΔiraP ΔdsrA strains containing pControl or pDksA_N88I as indicated. The bands were quantified and normalized relative to WT pControl (the error bars indicate means and SD; n = 3).

DksA_N88I activates transcription of the small regulatory RNA DsrA in vivo. (A) β-Galactosidase activity from a P_rpoS750-lacZ translational fusion in the WT (solid symbols) or a ΔdsrA mutant (open symbols) containing either pControl or pDksA_N88I. (B) P_dsrA-lacZ β-Gal activity plotted against increasing OD₆₀₀ in WT background. (C) P_arcZ-lacZ β-Gal activity plotted against increasing OD₆₀₀ in WT background. The curves shown are representative of at least 3 independent experiments.

Stimulation of P_iraP and P_dsrA promoter activity by DksA_WT or DksA_N88I in vitro with or without ppGpp. (A) Representative gel image illustrating fold activation by DksA with or without ppGpp on the dsrA promoter. Transcription was normalized to that in the absence of any factor (lanes 1 and 2), and fold activation for each reaction is shown beneath each lane (average of 2 reactions). Multiple-round in vitro transcription was performed with 10 nM RNAP and 170 mM NaCl at room temperature. Lanes 1 and 2, buffer only; lanes 3 and 4, 2 μM WT DksA only; lanes 5 to 11, 2 μM WT DksA plus ppGpp. The wedge indicates increasing ppGpp, from 3 to 200 μM. Plasmid templates also contained the RNA-1 promoter. Transcripts are indicated by arrows. (B) Representative gel image of transcription from the dsrA promoter as in panel A but with 0.2 μM or 2 μM DksA_N88I, as indicated. (C) Maximum activation observed from data sets illustrated in panels A and B plotted relative to transcription in the absence of either DksA or ppGpp. Shown is fold activation with 2 μM DksA_WT alone or with DksA_WT plus 100 μM ppGpp (black bars), 0.2 μM DksA_N88I alone or 0.2 μM DksA_N88I plus 25 μM ppGpp (light-gray bars), and 2 μM DksA_N88I alone or 2 μM DksA_N88I plus 6.25 μM ppGpp (dark-gray bars). The error bars indicate means and SD or range from at least two independent experiments. (D) Same as panel A except the template contained the iraP promoter. (E) Same as panel B except the template contained the iraP promoter. (F) Maximum activation of the iraP promoter from the data sets illustrated in panels D and E plotted relative to transcription in the absence of either DksA or ppGpp. Black bars, 2 μM DksA_WT alone or DksA_WT plus 100 μM ppGpp; light-gray bars, 0.2 μM DksA_N88I alone or 0.2 μM DksA_N88I plus 12.5 μM ppGpp; dark-gray bars, 2 μM DksA_N88I alone or 2 μM DksA_N88I plus 12.5 μM ppGpp. (G and H) Same as panels B and E except the DksA_N88I concentration was varied from 4 nM to 4 μM and no ppGpp was included in the reaction mixture (n = 3).

DksA_N88I and TraR stimulate the dsrA and iraP promoters in the absence of ppGpp in vivo. Overnight cultures were diluted 1:100 in LB medium at 32°C with 0.1 mM IPTG to induce DksA_N88I or TraR expression. (A to E) The activities of P_iraP-lacZ, P_dsrA-lacZ, or P_katE-lacZ fusions were monitored by measuring β-Gal activity during exponential growth at the optical densities shown on the x axes. The cells contained pControl, pDksA_N88I, or pTraR. Experiments were performed three times, and representative data are shown. The ppGpp⁰ strain (ΔrelA ΔspoT) is incapable of synthesizing ppGpp. It was shown previously that TraR can function without ppGpp in vivo (35). (A) Effect of DksA_N88I on expression of the P_iraP-lacZ fusion. (B) Effect of DksA_N88I on expression of the P_dsrA-lacZ fusion. (C) Effect of TraR on expression of the P_dsrA-lacZ fusion. (D) Effect of TraR on expression of the P_iraP-lacZ fusion. (E) Effect of DksA or TraR on expression of the P_katE-lacZ fusion in a WT or ΔrpoS background (open triangles). (F) Western blotting was performed, and σ^S levels were quantified at different times after TraR induction in a WT or ΔdsrA ΔiraP background as described in Materials and Methods. The error bars indicate SD.