Morphology of poly-β-hydroxybutyrate granules

Lundgren, D. G.; Okamura, Keizo; Marchessault, R. H.

doi:10.1016/s0022-2836(68)80009-9

Cited by 144 publications

(79 citation statements)

References 13 publications

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“…Neither a typical membrane nor membrane-bound polymerization machinery (14) is required to explain the observations, and this model incorporates the recent findings that the polymer remains in an amorphous state in the native granule (1,3,4,6,10,22,23). There are insufficient data to J. BAcTERiOL.…”

Section: * Corresponding Authormentioning

confidence: 93%

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Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

et al. 1993

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Antibodies raised against the Alcaligenes eutrophus poly-1-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules. The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation. From these basic observations and data from the recent literature, a model of granule assembly is proposed.Poly-p-hydroxybutyrate (PHB) accumulates as granular inclusions in the cells of a large number of bacterial species grown under conditions of nutrient limitation (2). In both Zoogloea ramigera (8,9,15,16,25,27,28) and Alcaligenes eutrophus (19,20), the condensation of two acetyl coenzyme A (CoA) units to form acetoacetyl-CoA by the enzyme 3-ketothiolase is followed by a stereospecific reduction step catalyzed by an NADP+-specific acetoacetyl-CoA reductase to form D-3-hydroxybutyryl-CoA, the substrate for PHB synthase. Both the 13-ketothiolase and acetoacetyl-CoA reductase are soluble cytosolic enzymes, whereas the PHB synthase is found attached to the PHB granules.Purified granules contain around 98% PHB by weight, with the remainder being made up of proteins and lipids (17,22). It was previously concluded that the physical state of the polymer inside the granules in vivo was that of a crystalline solid, similar to that of the extracted polymer (12,14,21). More recently, nuclear magnetic resonance studies of whole cells have provided the first evidence that the "granule" is in fact in a mobile, amorphous state in vivo (3,4). Both the presence of plasticizers including water (4, 18) or lipid (22) and the kinetics of the crystallization process (6, 10) have been proposed to describe how the granules are maintained in the amorphous state inside the cell. Additional studies have concluded that the PHB granules inside the cell are surrounded by a membrane containing the synthase enzyme and that the D-3-hydroxybutyryl-CoA monomers are transported across the membrane in conjunction with polymerization (5, 13, 24, 32).Our current research activities are focused on understanding the role of the PHB synthase in the biogenesis of the PHB granules in A. eutrophus. In this regard there are two main issues: (i) the mechanism by which the PHB synthase initiates and carries out the condensation of literally thousands of D-3-hydroxybutyryl-CoA units to form the polymer chains and (ii) the role of this mechanism and the PHB synthase protein in the assembly of the PHB granules. We report here the immunocytochemical localization of the PHB synthase on the surface of the granules and describe a working hypothesis for granule formation. * Corresponding author.Preparation and analysis of anti-PHB synthase antibodies. In order to obtain purified A. eutrophus PHB synthase protein for the purpose of preparing antibodies, the C-terminal region of the phbC gene from plasmid pAeT42 encoding residues 86 to 589 of the PHB synthase (26) was ...

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Section: * Corresponding Authormentioning

confidence: 93%

“…It was previously concluded that the physical state of the polymer inside the granules in vivo was that of a crystalline solid, similar to that of the extracted polymer (12,14,21). More recently, nuclear magnetic resonance studies of whole cells have provided the first evidence that the "granule" is in fact in a mobile, amorphous state in vivo (3,4).…”

mentioning

confidence: 87%

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

et al. 1993

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“…The primary structure of the putative ORF1 gene product did not display significant similarity to any other protein sequences in the database. If ORF1 is expressed, its gene product may possess a function similar to that of small proteins identified at the surface of PHA granules in other bacterial species (12,13,24). Northern blot analysis.…”

Section: Resultsmentioning

confidence: 99%

Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes

1995

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The polyhydroxyalkanoic acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaA Ac (encoding a ␤-ketothiolase), phaB Ac (encoding an acetoacetyl coenzyme A reductase), and phaC Ac (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly-␤-hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaB Ac , an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.Polyhydroxyalkanoic acids (PHAs) are a family of bacterial polyesters synthesized by a wide range of organisms under conditions of nutrient limitation in the presence of an excess carbon and energy source (1). These polymers have attracted significant attention for their potential commercial exploitation as biodegradable plastics (22). The most abundant and best-studied PHA is poly-␤-hydroxybutyrate (PHB), a linear, unbranched homopolymer built up of (R)-3-hydroxybutyric acid units.The physiology and molecular genetics of PHB biosynthesis have been extensively studied in Alcaligenes eutrophus (33). More recently, genes involved in PHA metabolism have also been cloned from numerous other organisms (see the review in reference 32). In most organisms, PHB is synthesized via a three-step pathway which involves the condensation of two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetylCoA via a ␤-ketothiolase (PhaA), reduction of acetoacetylCoA to D-(Ϫ)-␤-hydroxybutyryl-CoA via an NADPH-dependent acetoacetyl-CoA reductase (PhaB), and polymerization to PHB via a PHA synthase (PhaC) (33).Recently, the PHA synthase gene was cloned from an Acinetobacter strain isolated from a modified activated sludge treatment plant (31). Acinetobacter species have been suggested to be the major organisms responsible for enhanced biological phosphate removal from wastewater treated in alternating anaerobic/aerobic activated sludge systems (5). Biochemical models of this overall process involve the breakdown of polyphosphate and synthesis of PHB during the anaerobic stage and breakdown of PHB and production of polyphosphate in the subsequent aerobic stage (8). Strains of Acinetobacter which can accumulate up to 10% phosphorus (10) and 15% PHB (28) on a dry weight basis have been isolated.In A. eutrophus, all three PHA biosynthetic enzymes are synthesized constitutiv...

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“…At the very beginning, the PHA synthase protein with the growing polyester chain may form micromicelles with nascent hydrophobic PHA chains attached by hydrophobic interactions in the center and PHA synthase proteins at the surface, giving rise to nascent inclusion bodies. It may be that, at this stage, the boundary layer at the surface of the inclusion body is mainly or even exclusively composed of PHA synthase protein, as proposed by Ellar et al (6). When the inclusion bodies become larger, the composition of the boundary layer obviously changes and, besides the PHA synthase protein, other components such as phospholipids (10) and phasins (for review, see 21) are inserted.…”

Section: And Discussionmentioning

confidence: 96%

“…The process of inclusion body formation exhibits analogies to an emulsion polymerization process (4,6). The emulsion of hydrophobic PHA molecules in the predominantly hydrophilic cytoplasm is obviously stabilized by proteins and phospholipids.…”

Section: And Discussionmentioning

confidence: 99%

Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies.

Mayer

Madkour

Pieper‐Fürst

et al. 1996

J. Gen. Appl. Microbiol.

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The boundary layer of PHA inclusion bodies isolated from Chromatium vinosum, Pseudomonas oleovorans, and Rhodococcus Tuber was investigated by negative staining and transmission electron microscopy. A typical boundary layer exhibits a basic lattice composed of regularly arranged phasin molecules and phospholipids, forming a thin, nevertheless mechanically stable cover surrounding the content of the inclusion body. Depending on the bacterial strain under investigation, the lattice parameters of the boundary layer may vary. Usually, values between 3.3 and 9.6 nm are observed for the spacing, and the lattice is rectangular. Enzyme particles interpreted as PHA synthase particles are attached to, or inserted into the basic lattice. They cover not more than 20% of the total surface of the inclusion body.These enzyme particles measure between 8 and 12 nm in diameter, are made up of subunits and occur as single units or small aggregates. No indications have been obtained which would support the view that the boundary layer is a double-layer of proteins with phospholipids in between.Rather, a visual inspection of detached boundary layers revealed that the boundary layer is a monolayer exhibiting only one kind of basic lattice. In regions where this monolayer had been artificially removed from the inclusion body, the surface of the contents of the inclusion body was exposed.

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Morphology of poly-β-hydroxybutyrate granules

Cited by 144 publications

References 13 publications

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes

Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies.

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