Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber

Pieper‐Fürst, Ursula; Madkour, Mohamed Hussein; Mayer, Frank; Steinbüchel, Alexander

doi:10.1128/jb.176.14.4328-4337.1994

Cited by 105 publications

(99 citation statements)

References 38 publications

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“…These findings support the view that phasins are the major component of the boundary layer of a mature inclusion body. The properties of the phasin molecules, as demonstrated recently (15,16,25), indicate that these molecules, due to their amphiphilic character, separate hydrophilic compartments (the cell cytoplasm) from hydrophobic compartments (the contents of the PHA inclusion body), and that the phasins form a monolayer. This conclusion is supported by the results of a visual inspection of artificially detached and shifted boundary layers as depicted in this investigation; only one layer of the basic lattice is visible under these conditions, and after artificial removal of this single layer, the contents of the inclusion body are exposed.…”

Section: And Discussionmentioning

confidence: 99%

“…The samples were negatively stained with uranyl acetate, and depicted as described previously (15). Electron micrographs were obtained at calibrated magnifications.…”

Section: Methodsmentioning

confidence: 99%

“…When isolated PHA granules are subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the electropherograms revealed low molecularweight proteins as the predominant protein species in almost all PHA-accumulating bacteria (7,12,14,19,25,26). Based on the amino-acid sequence data of phasin molecules, models of the structure and composition of the PHA granule surface in Rhodococcus ruber and for the anchoring of the phasin (the GA 14 protein) monomer in a phospholipid monolayer were postulated (15). The region of the GA 14 protein, which is responsible for the binding of this phasin to the surface of the PHA granule, was identified (16).…”

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confidence: 99%

“…In a first attempt, we investigated three such systems (isolated PHA inclusion bodies obtained from C. vinosum, P. oleovorans, and R. Tuber, respectively) by the application of conventional negative staining procedures and transmission electron microscopy. In previous experiments, aspects of the molecular architectures of the PHA inclusion bodies of these bacteria were investigated with immunoelectron microscopic techniques (13,(15)(16)(17)21,23,25).…”

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confidence: 99%

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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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Section: And Discussionmentioning

confidence: 99%

“…The samples were negatively stained with uranyl acetate, and depicted as described previously (15). Electron micrographs were obtained at calibrated magnifications.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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 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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Poly(3‐Hydroxyalkanoates)

Kessler¹

1999

Encyclopedia of Bioprocess Technology

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Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber

Abstract: The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated 500 protein of Rhodococcus ruber (the GA14 protein) was analyzed.

Cited by 105 publications

References 38 publications

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

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

Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes

Poly(3‐Hydroxyalkanoates)

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