Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

Pfeiffer, Daniel; Jendrossek, Dieter

doi:10.1128/jb.00779-12

Cited by 81 publications

(73 citation statements)

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“…This finding is in agreement with previous contributions of others (41). In our earlier studies on PHB granule formation, we showed by fluorescence microscopy that newly synthesized PHB granules occurred very rapidly within 5 min after application of PHB permissive conditions in starved (PHB-free) cells of R. eutropha (i.e., after transfer of starved cells to fresh NB-gluconate medium) (24). If a PHB granule of ϳ250 nm in diameter is formed in the cells within only 5 min, it is unlikely that PHB synthase has a significant lag phase of 2 to 4 min in vivo.…”

Section: Resultssupporting

confidence: 81%

“…The micelle model was the favorite model for many PHA researchers in the past. However, this model cannot explain the nonrandom localization of "early" PHB granules, i.e., the frequent observation of PHB granules near the cell center in nongrowing cells (46)(47)(48) or the finding of peripherylocated PHB granules in growing cells (24,45,49). Nucleoid occlusion of PHB granules (50) in growing cells might be one explanation for the frequent observation of periphery-localized PHB granules, and this assumption would allow to keep the micelle model.…”

Section: Discussionmentioning

confidence: 99%

“…Nineteen proteins apparently are associated with the proteinaceous surface layer of PHB granules in R. eutropha H16. PHB granule-associated proteins (PGAPs) of R. eutropha H16 include (i) PHB synthase PhaC1 (12)(13)(14), (ii) seven PHB depolymerases (PhaZs, PhaZa1 to PhaZa5, PhaZd1, and PhaZd2) (15)(16)(17)(18), (iii) two PHB oligomer hydrolases (PhaZb and PhaZc [alternative designations PhaY1 and PhaY2]) (19,20), (iv) seven phasins (PhaP1 to PhaP7) (21)(22)(23)(24), and (v) two DNA-and PHB-binding proteins (PhaR and PhaM) (25)(26)(27)(28). At present, it is not known which and how many of these individually identified PGAPs are expressed and localized on a PHB granule at a given time in vivo.…”

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confidence: 99%

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PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Pfeiffer

Jendrossek

2014

Appl Environ Microbiol

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Pfeiffer

Jendrossek

2014

Appl Environ Microbiol

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“…Only the proteins that colocalize with PHB granules are true in vivo PGAPs. This method has been used to confirm the subcellular localization of PHB synthases, phasins, and PHB depolymerases (32)(33)(34)(35)(36)(37)(38)(39). Because of the high number (Ͼ400) of proteins identified in the PHB granule fraction by proteome analysis (29), the in vivo confirmation of their subcellular localizations would require the construction of hundreds of fusions with green fluorescent protein.…”

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confidence: 99%

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Sznajder

Pfeiffer

Jendrossek

2015

Appl Environ Microbiol

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Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative ␣/␤-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. P olyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHA) are storage compounds for carbon and energy and are widespread in prokaryotic species (1). PHB is deposited in the form of 200-to 500-nm particles (granules) when PHB-accumulating bacteria are cultivated in the presence of a surplus of a suitable carbon source. Ralstonia eutropha H16 (alternative designation, Cupriavidus necator) has become the model organism of PHB research, and the species is also used in industrial processes to produce PHB as a biodegradable polymer with plastic-like properties (2-4). The intensive research of the last few decades has led to a good understanding of the biochemical routes leading to PHB/PHA and of the biochemical properties of proteins with established functions in PHB/PHA metabolism. For example, the key enzymes of PHB synthesis, the PHB synthases (PhaCs), of many PHB-accumulating species have been purified; the coding genes were cloned; and the polymerization reaction has been studied (for overviews and recent results, see references 5 to 11). Meanwhile, much evidence has accumulated showing that PHB granules are not simply storage molecules but represent well-defined subcellular organelles that consist of a polymer core and a surface layer to which many proteins with specific functions are attached (12). Besides the aforementioned PHB synthase, small amphiphilic polypeptides, so-called phasin proteins (PhaP...

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“…Later on, six other phasins (PhaP2 to PhaP7) were detected, and all of them were detected at the surface of PHB granules during in vivo or in vitro binding studies. However, the amount of PhaP1 at the surface of PHB granules is by far the largest in comparison to the amounts of other phasins (15)(16)(17). The phasins coat the PHB granules, but a catalytic function remains unknown so far.…”

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confidence: 99%

Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

Eggers

Steinbüchel

2013

Journal of Bacteriology

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bDegradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ⌬phaP1 and ⌬phaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ⌬phaP1 and ⌬phaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the ␤-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated. P oly(3-hydroxybutyrate) (PHB) is a short-carbon-chainlength polyester belonging to the bacterial polyhydroxyalkanoates (PHAs), which are formed by a wide range of Bacteria and Archaea and comprise a large variety of constituents (1-3). PHAs are stored as granules in the cytoplasm when one nutrient is limited and a carbon source is provided in excess. They serve as carbon and energy storage compounds and are degraded if no carbon source is available and if the limiting element is provided again (4). Due to their thermoplastic properties and biodegradability, PHAs are of great interest for industry (5-7).The model organism for PHB metabolism is the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha H16, which was isolated in 1961 (8-10). About 50 years of research unraveled many aspects of the synthesis and composition of PHB. T...

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Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

Cited by 81 publications

References 56 publications

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

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