Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Abstract

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-

Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 of Ralstonia eutropha Are Phosphorylated In Vivo

“…In a previous study, we assumed that a combination of allosteric regulation and covalent modification of the enzymes involved in PHB accumulation and mobilization is necessary to regulate the PHB content of R. eutropha. The PHB content of R. eutropha was previously shown to be dependent on the concentration of alarmones such as ppGpp (31). The presence of phosphorylations would be in line with previous data.…”

“…No phosphorylated peptides could be identified in samples harvested at the exponential growth phase or from cells grown on mineral medium with 2% fructose. As shown in previous studies, R. eutropha does not degrade PHB under this condition (24,31). Phosphorylated peptides of the major PHB depolymerase, PhaZa1, were identified in samples at all stages of growth and on different media.…”

“…This expression pattern poses the questions of how the synthesis and mobilization of carbonosomes are regulated and how a futile cycle of simultaneous synthesis and degradation is avoided. One possibility is that PHA synthesis and degradation are controlled by signaling molecules as described in a previous study (31). An additional possibility could be through allosteric regulation of PHB synthase and PHB depolymerase activities.…”

“…The fluorophore eYFP was detected with the aid of a standard filter set (excitation, 500/24 nm; emission, 542/27 nm) as previously described (6). PHB granules were visualized by fluorescence microscopy of Nile red-stained cells using standard filters (excitation, 562/40 nm; emission, 594 nm [longpass filter]) as described previously (6,31). Insoluble inclusions could also be visualized by bright-field microscopy and appeared as dark spheres.…”

Inducible expression of (pp)pGpp synthetases inStaphylococcus aureusis associated with activation of stress response genes

“…Nucleotids were isolated based on published protocol (Juengert et al, 2017). Briefly, strains were 411 grown in CDM overnight, diluted to an OD600 = 0.05 and grown in CDM until an OD600 = 0.3.…”