In this study, we constructed a set of single-, double-, and triple-gene deletion strains of Ralstonia eutropha H16 in (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2) and polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) genes and determined the impact on the levels of (p)ppGpp and on accumulated PHB. Double deletion mutants in both 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 determined in strains in which the concentration of (p)ppGpp was increased by the overexpression of (p)ppGpp synthase (SpoT2) and absence of (p)ppGpp hydrolase. Determinations of (p)ppGpp levels in wild type R. eutropha under different growth conditions and induction of stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of remaining PHB in later growth phases by influencing the efficiency of the PHB mobilization system in the stationary growth. Data on a previously constructed ΔspoT2 strain (Brigham J, Speth DR, Rha C, Sinskey AJ. 2012. AEM 78:8033-44) were identified as an experimental error in strain construction and we retract the previous statement that the spoT2 gene product is essential for PHB accumulation in R. eutrophaImportance 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 over the last three decades in several laboratories 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 to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that stringent response in Rhizobium etli and in R. eutropha is involved in 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 an 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 massive over-production of PHB. Contrary, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.
This research output has an Altmetric Attention Score of 7. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 24 June 2025.
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