Poly-(
R)-3-hydroxybutyrate (PHB) homeostasis in
Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore
intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process
requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative
Western blotting were carried out on batch cultures of
R. eutropha H16 in order to gain insight into how expression of the PHB-related genes
phaA,
phaB,
phaC,
phaP,
phaR,
phaZ1a,
phaZ1b, and
phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase.
phaA,
phaB,
phaC,
phaR, and
phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was
shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during
PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of
phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation
did not depend on PHB production as in the case of
phaP. No evidence of
phaZ1c expression was found under the experimental conditions used in this study.
Lawrence and Schoenheit made equal contributions to this work