Fig. 5. Alanine substitutions in the H-loop leads to increased (p)ppGpp synthesis.

a Primary sequence of the H-loop region (indicated within a box) of RelA. Arrows indicate positions which have been substituted to alanine in RelA^QUAD (R117A, Q118A, K120A and H123A). b Functional assay of RelA^QUAD. Cell cultures of MG1655 relA::HTF, MG1655 ΔrelA and MG1655 relA^QUAD::HTF, were washed in PBS, serial diluted and spotted onto MOPS MM SMG plates (SMG resistance). The plates were incubated ON at 30 °C (See Supplementary Fig. 1f for loading controls). c (p)ppGpp measurements of strains in b). Cells were grown exponentially in MOPS minimal medium containing ³²P-labeled phosphate. Isoleucine starvation was induced by addition of L-valine, to a final concentration of 500 μg/mL. Samples were collected before (time zero) and after starvation, precipitated and separated by thin layer chromatography. The RelA^QUAD::HTF curve with black crosses is based on the average of n = 2 biological independent samples see Supplementary Fig. 1m and 1n. d Representative TLC from a, positions of GTP, ppGpp and pppGpp are indicated.