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

Abstract

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 ...

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Granules membrane coat is about 2 nm thick, containing 0.5% lipid and 2% protein, of the granule weight (138). PHA polymerases are present in the cytosol; however, they are only active when localized on the surface of granules (103,132,134). The intracellular depolymerases are required for mobilization of the reserve polyester and are attached to the granule surface (139).…”

Efficient P(3HB) extraction from Burkholderia sacchari cells using non-chlorinated solvents

“…The purity of the recovered polymers was assessed, as cellular impurities such as proteins [35] [36] and traces of solvent can be present, even though solvent remains were minimized by carefully drying the samples. Table 3 reports the calculated purity of each sample.…”

Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism

“…In R. eutropha H16, the key component of PHB biosynthesis is the PHB synthase encoded by phaC1. The gene is constitutively expressed, and the enzyme occurs as a soluble protein only at the beginning of PHB biosynthesis; upon onset of PHB biosynthesis, the enzyme becomes granule-bound (Haywood et al, 1989;Gerngross et al, 1993). The constitutive expression of phaC1 is affirmed by the first K. Peplinski and others two transcription analyses presented in this study, comparing (i) transcriptomes from exponential growth phase with transcriptomes from the transition phase and (ii) transcriptomes from transition phase with transcriptomes from the stationary growth phase of R. eutropha H16.…”