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Updated: Feb 1, 2026

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain
Séverin Ronneau1, Julien Caballero-Montes2, Jérôme Coppine1
1Bacterial Cell cycle & Development (BCcD), Biology of Microorganisms Research Unit (URBM), Namur Research Institute for Life Science (NARILIS), University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium.
Abstract:
Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA) domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp synthetase/hydrolase of Caulobacter crescentus. We show that SpoT requires the ACT domain to efficiently hydrolyze (p)ppGpp. In addition, our in vivo and in vitro data show that the phosphorylated version of EIIANtr (EIIANtr∼P) interacts directly with the ACT and inhibits the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr∼P and SpoT/Rel along with the phosphotransferase system (PTSNtr)-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, this work suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)ppGpp levels in response to nitrogen availability.
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