Figure 4.

In vitro activity characterization of the putative (p)ppGpp synthetases RelP^*_Cg and RelS_Cg, as well as the enzyme Rel_Cg. Graphical representation of the HPLC analysis of 50 μL assay reactions containing 500 ng of the corresponding enzyme and ATP+GTP (A), ATP+GDP (B) and ATP+GMP (C) as substrate combinations with a concentration of 4 mM each. HPLC separation was performed using a SeQuant ZIC-pHILIC column and isocratic elution with 38% of 10 mM ammonium bicarbonate buffer (pH 9.3) and 62% acetonitrile. The reaction products were identified by their specific masses and retention times. Their relative abundance is given as the peak area of the UV signal (252 nm) per mg of the respective enzyme per hour. The results shown were corrected by the values determined for enzyme-free controls. For some measurements pppGpp (^*) or pGpp (^**) were detected by their characteristic MS signals, but the UV detection limits were not reached.