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. 2018 Nov 29;47(2):843–854. doi: 10.1093/nar/gky1201

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — The ACT domain is required in vivo to support the hydrolase activity of SpoT. (A and B) Absence of the ACT domain of SpoT promotes G1 accumulation and decreases growth rate upon artificial exogenous production of (p)ppGpp. (A) Flow cytometry analysis to determine DNA content in asynchronous population of WT (RH50), spoT_Y323A(RH1844), spoT_D81G_Y323A(RH2193), ΔptsP spoT_Y323A (RH2196), spoT _Y323A_ΔACT(RH1586) and ΔptsP spoT_Y323A_ΔACT(RH2492) strains with (black bars) or without (grey bars) P_xylX::relA-FLAG grown for 6 h in PYE medium supplemented with 0.1% of xylose. The flow cytometry data were normalized to the WT without P_xylX::relA-FLAG (100%). (B) The growth of strains used in (A) was measured for 24 h in PYE medium supplemented with 0.1% of xylose. Error bars = SD, n = 3. (C) The regulatory ACT domain of SpoT is required in vivo to degrade (p)ppGpp in nitrogen-replete condition (+N). The intracellular levels of (p)ppGpp were evaluated by TLC after nucleotides extraction from spoT_Y323A(RH1844), spoT_D81G_Y323A(RH2193), ΔptsP spoT_Y323A (RH2196), spoT_Y323AΔ_ACT(RH1586) and ΔptsP spoT_Y323A_Δ_ACT(RH2492) strains harbouring P_xylX::relA-FLAG and grown for 2 h in glutamine (Q) containing media (+N) supplemented with 0.1% xylose.