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In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/ 10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a highperformance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and mas-

In this study, we screened PHB synthase PhaC1 and PHB depolymerase PhaZa1 of for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phase. Thr373 of PHB synthase PhaC1 was phosphorylated in the stationary growth phase but was not modified in the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in phosphorylated form both in the exponential and in the stationary growth phase. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modification of Thr26 and Ser35 of PhaZa1 reduced the ability of to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for modulation of the activities of PHB synthase and PHB depolymerase. Polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and other stress conditions. The simultaneous presence of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in (alternative designation) has been previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of identified residues. Furthermore, we conducted and analysis of PHB synthase activity and PHB contents.