Abstract
Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).
Highlights
Various microorganisms accumulate polyhydroxyalkanoates (PHAs) when carbon and energy sources are readily available
PHB granules formation by P. denitrificans cells using confocal microscopy The lipophilic core of PHB granules is covered by a monolayer of phospholipids (Steinbüchel et al 1995); only appropriate lipophilic dyes can be used to stain it (Vida and Emr 1995)
P. denitrificans cells growing at the mid-exponential phase of growth were stained with different membrane dyes, including: MitoTrackerTM Green FM (MTG), FM4-64 and Nile Red, and analyzed by laser scanning confocal microscopy (LSCM)
Summary
Various microorganisms accumulate polyhydroxyalkanoates (PHAs) when carbon and energy sources are readily available. These polymers serve as reserves of carbon and energy to allow cell survival during starved. There are controversial results on the specific subcellular localization of the PHB granules in distinct bacterial models. The investigation of PHB synthesis in P. denitrificans by both microscopy and biochemical analyses to study pattern formation of the granules. The rationale was that GSMMs are comprehensive compilations of all metabolic reactions taking place in a particular cell, and as such can be used to predict theoretical yields of a bacterium during growth on different carbon and energy sources. In this study we investigated the formation of PHB granules and subcellular localization in P. denitrificans, a denitrifying model organism with a well-documented ability to accumulate PHB
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