Fig. 3. pGpp is produced by NahA in vivo and has a distinct binding spectrum compared to (p)ppGpp.

a LC-MS analyses of pGpp, ppGpp, and pppGpp of wild type and ΔnahA in log phase and stationary phase. Normalized ion count is ion count per OD_600nm per unit volume of the culture. Error bars represent standard errors of the mean of three biological replicates. A two-tailed two-sample equal-variance Student’s t test was performed between samples indicated by asterisks. Asterisks indicate statistical significance of differences (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). b In vitro hydrolysis of pGpp, ppGpp, and pppGpp by B. subtilis Rel. For each nucleotide, 200 nM Rel was incubated with a mix of 100 μM unlabeled alarmone and a small amount of 5′-α-³²P-labeled version. The degradation of the alarmone was analyzed by TLC to measure the decreased radioactivity. Error bars represent standard errors of the mean of three replicates. c DRaCALA of purified His-MBP-tagged B. anthracis proteins (1 μM) with <0.1 nM of identical amount of 5′-α-³²P-labeled pGpp, ppGpp, and pppGpp. d Quantification of DRaCALA in c. Error bars represent standard errors of the mean of three replicates except for control group (six replicates). e HprT enzymatic activities in the presence of indicated concentrations of pGpp, ppGpp, and pppGpp. The reaction was performed with 20 nM HPRT, 1 mM PRPP, and 50 μM guanine¹⁴. Error bars represent standard errors of the mean from three replicates. f–h Titrations of B. anthracis GTPases and quantification of their binding to pGpp, ppGpp and pppGpp using DRaCALA: GTPases HflX (f), Obg (g), and translation elongation factor G (EF-G) (h). Error bars represent standard errors of the mean of three replicates. i Schematic showing the relationship between pGpp-binding targets and (p)ppGpp-binding targets.