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. 2016 Apr 25;7:11423. doi: 10.1038/ncomms11423

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(a) spoT_D81G is insensitive to the absence of EIIA^Ntr. G1 proportion was measured in wild-type (WT; RH50), spoT_D81G (RH1752), spoT_D81G ΔptsP (RH1727), spoT_D81G ΔptsH (RH2013), spoT_D81G ΔptsN (RH1999), spoT_D81G ΔptsN ΔptsP (RH2014) and spoT_D81G ΔptsN ΔptsH (RH2015) grown in complex media (PYE) and normalized to the WT (100%). Error bars=s.d.; n=3. (b) The hydrolase activity of SpoT is required for growth on an artificial exogenous production of (p)ppGpp. Growth of spoT_Y323A, spoT_{D81G Y323A}, ptsN_H66AspoT_Y323A, ptsN_H66EspoT_Y323A and ΔptsP spoT_Y323A expressing a truncated version of E. coli relA from the inducible xylX promoter (PxylX::relA-FLAG) on PYE medium supplemented with 0.1% of xylose. (c) Reduction of SpoT hydrolase activity led to a G1 extension on an artificial exogenous production of (p)ppGpp. Flow cytometry analysis to determine DNA content in asynchronous population of WT, spoT_Y323A, spoT_{D81G Y323A}, ptsN_H66AspoT_Y323A, ptsN_H66EspoT_Y323A and ΔptsP spoT_Y323A with (dark grey bars) or without (light grey bars) PxylX::relA-FLAG in PYE medium supplemented with 0.1% of xylose. The data were normalized to the WT without PxylX::relA-FLAG (100%). Error bars=s.d.; n=3. (d) The hydrolase activity of SpoT is required to degrade (p)ppGpp in +N condition. Intracellular levels of (p)ppGpp detected by TLC after nucleotides extraction of spoT_Y323A, spoT_{D81G Y323A}, ptsN_H66AspoT_Y323A and ΔptsP spoT_Y323A containing PxylX::relA-FLAG in +N or −N medium supplemented with 0.1% xylose.