Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha

The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, "frozen-hydrated" state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts.