Implication of Rsd during a carbon source downshift. Growth curves were measured during a carbon downshift from glucose to casamino acids. Cells grown overnight in LB medium were harvested, washed, suspended in MOPS minimal medium containing 0.1% casamino acids and 0.02% glucose. Then, 100-μL aliquots of each strain were transferred into a 96-well plate and growth was monitored at 600 nm using a multimode microplate reader (TECAN). The mean and standard deviation of three measurements are shown. In the presence of glucose, HPr is predominantly dephosphorylated and then sequesters Rsd from SpoT to maintain the (p)ppGpp hydrolytic activity at basal levels. On depletion of glucose, however, the ppGpp level abruptly increases in relA+ strains. Therefore, the (p)ppGpp hydrolase activity of SpoT needs to be stimulated to counterbalance the RelA-dependent large accumulation of (p)ppGpp. This appears to be achieved by phosphorylation of HPr. Because phospho-HPr cannot interact with Rsd, Rsd can bind and activate the (p)ppGpp hydrolase activity of SpoT to balance the intracellular (p)ppGpp concentrations, which contributes to the growth resumption of E. coli cell during a carbon downshift.