Calcium Involved Directional Organization of Polymer Chains in Polyester Nanogranules in Bacterial Cells

Abstract

Soil bacteria accumulate polyesters (typically poly([R]-3-hydroxybutyrate (PHB), in which one end of the chain terminates with a carboxyl group) in the form of hydrated, amorphous nanogranules in cells. However, it is not clear what drives the structure of these biomaterials inside bacterial cells. Here, we determined that calcium guides intracellular formation of PHB nanogranules. Our systematic study using the surface zeta potential measurement and the carboxyl-specific SYTO-62 dye binding assay showed that the terminal carboxyl is not exposed to the granule surface but is buried inside native "unit-granules" comprising the mature granule. Extracellular Ca2+ was found to mediate the formation of these PHB unit-granules, with uptaken Ca2+ stored inside the granules. Comparative [Ca2+]-dependent fluorescence spectroscopy revealed that the native granules in Cupriavidus necator H16 act as a Ca2+ storage system, presumably for the regulation of its cytosolic Ca2+ level, but those from recombinant Escherichia coli do not. This study reveals intimate links between Ca2+ and native granule formation, and establishes a novel mechanism that intracellular PHB granules function as Ca2+ storage in order to relieve soil bacteria from Ca2+ stress.