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Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16.

ABSTRACT: Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

SUBMITTER: Kobayashi T

PROVIDER: S-EPMC1196019 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16.

Kobayashi Teruyuki T Uchino Keiichi K Abe Tomoko T Yamazaki Yuya Y Saito Terumi T

Journal of bacteriology 20050801 15

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount ...[more]

PMID: 16030206

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Project description: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. 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 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 high-performance 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 massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Dataset Information

Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16.

Publications

Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16.

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