FIG 3.

In vivo and in vitro evidence for (p)ppGpp synthetic activities of RelP and RelQ. (A) Detection of (p)ppGpp synthesis after amino acid deprivation in vivo. ³²P-labeled nucleotides of formic acid extracts of S. aureus were detected by thin-layer chromatography. Strain HG001 (WT), the rsh_syn mutant, and the rsh_syn relP relQ [(p)ppGpp⁰] triple mutant were grown to the exponential phase, followed by the addition of mupirocin (0.5 μg/ml) for 20 min. (B) Characterization of the (p)ppGpp synthase activities of RelP and RelQ in vitro. Purified RelP or RelQ proteins (0.5 μg) were assayed for (p)ppGpp synthase activity in the presence of [γ-³²P]ATP, 2 mM ATP, and either 2 mM GTP or 2 mM GDP. Reaction mixtures were analyzed by 1D thin-layer chromatography. The positions of the origin and signals corresponding to pppGpp, ppGpp, and ATP are indicated.