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

The boundary layer of PHA inclusion bodies isolated from Chromatium vinosum, Pseudomonas oleovorans, and Rhodococcus ruber 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.