(A) pppGpp levels increase concomitantly with increased GTP levels in WT cells following guanosine treatment (Guo). 32P-labeled WT cells were treated with 1 mM Guo. At the indicated times after treatment, nucleotides were extracted, measured by TLC, and normalized to T=0.
(B) Schematic of a negative feedback loop of GTP synthesis with pppGpp.
(C) Two-dimensional TLC of 32P-labeled nucleotides with ATP and GTP indicated. WT (left panels) and (p)ppGpp0 (right panels) cells before (upper panels) and 20 minutes after addition of 1 mM Guo (lower panels) are shown.
(D–E) Quantification of GTP (D) and ATP (E) levels by LC-MS/MS of WT (red) and (p)ppGpp0 (blue) cells in a time course after treatment with 1 mM Guo, normalized to T=0.