Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Abstract

Atomic force microscopy analysis of polyhydroxyalkanoate (PHA) inclusions isolated from sonicated Ralstonia eutropha cells revealed that they exhibit two types of surface structure and shape; rough and ovoid, or smooth and spherical. Smooth inclusions possessed linear surface structures that were in parallel arrays with 7-nm spacing. Occasionally, cracks or fissures could be seen on the surface of the rough inclusions, which allowed a measurement of approximately 4 nm for the thickness of the boundary layer. When the rough inclusions were imaged at higher resolution, globular structures, 35 nm in diameter, having a central pore could be seen. These globular structures were connected by a network of 4-nm-wide linear structures. When the inclusions were treated with sodium lauryl sulfate, the boundary layer of the inclusion deteriorated in a manner that would be consistent with a lipid envelope. When the boundary layer was largely gone, 35-nm globular disks could be imaged laying on the surface of the filter beside the inclusions. These data have facilitated the development of a preliminary model for PHA inclusion structure that is more advanced than previous models.

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

“…Additionally, there are many pores observed in the crystalline lamellar layer that do not have a corresponding globular structure over them. The strongest evidence for this supposition comes from the prior AFM study in which globular structures with central ϳ15-nm pores were observed at a high density under the surfaces of enveloped inclusions (4).…”

“…Cupriavidus necator H16 was grown overnight in a nutrient broth culture, and this culture was used to inoculate a 50-ml culture (in a 250-ml flask) such that the initial optical density at 600 nm was between 0.05 and 0.1. A supplemented minimal medium was used (24 4 -citrate, 0.10 ml SL6 trace elements (22), 5 ml 20% (wt/vol) Casamino Acids, 0.1 ml 0.5% (wt/vol) thiamine, and 0.4% (wt/vol) fructose. When cultures were incubated longer than 18 h, another aliquot of 0.4% fructose was added at approximately 20 h postinoculation.…”

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

“…However, electron microscopy studies showing membrane-like material surrounding PHA granules in intact cells (118) or isolated granules (119) provided evidence for the budding model. Tian et al showed that early stage granules are not randomly distributed in the cytoplasm and close to the inner cell membrane (120), as would be anticipated from the two models of granule formation (121).They found that emerging granules arose from only the center of the cell at unknown mediation elements suggesting a new model for PHA granule formation ( Figure 3) (120). Dennis et al observed large structures (35 nm) on the surface of PHB-containing granules from C. necator cells using atomic force microscopy which might function as synthesis-degradation centers (121).…”

“…Tian et al showed that early stage granules are not randomly distributed in the cytoplasm and close to the inner cell membrane (120), as would be anticipated from the two models of granule formation (121).They found that emerging granules arose from only the center of the cell at unknown mediation elements suggesting a new model for PHA granule formation ( Figure 3) (120). Dennis et al observed large structures (35 nm) on the surface of PHB-containing granules from C. necator cells using atomic force microscopy which might function as synthesis-degradation centers (121). Recent fluorescence microscopic studies employing green fluorescent protein (GFP)-labeled PHA synthase, that is, GFP, were fused to the N-terminus of class I and class II PHA synthases, without affecting PHA particle formation, enabled in vivo monitoring of PHA granule formation as well as subcellular localization (122).…”

Kinetic Studies of Polyhydroxybutyrate Granule Formation in Wautersia eutropha H16 by Transmission Electron Microscopy

“…Early electron microscopy studies of PHB granules from Bacillus cereus, Bacillus megaterium (23), Rhodospirillum rubrum (3), and Chlorogloea fristschii (19) all revealed an atypical membrane-like material surrounding the surface of granules, which varied in thickness from 3 nm to 20 nm depending on the species. Recent atomic force microscopy studies of granules freshly isolated from W. eutropha with minimum perturbation also demonstrated that there was a 3-to 4-nm-thick boundary layer surrounding the surface of the granules (7). In addition, globular structures, which were 35 nm in diameter with a central pore, were also reported to be on the surface of the granules and were proposed to be centers for PHB synthesis and depolymerization.…”