Three-dimensional label-free visualization and quantification of polyhydroxyalkanoates in individual bacterial cell in its native state

Significance Polyhydroxyalkanoates (PHAs) are bacterial polyesters considered as sustainable and environmentally friendly substitutes for petroleum-based plastics. Despite great advances in PHA research, spatiotemporal characteristics of PHA granule formation and distribution in a live bacterial cell are not well understood. Here, we report the results of a three-dimensional (3D) analysis of live bacterial cells accumulating poly(3-hydroxybutyrate) (PHB), the best-studied member of PHAs. Optical diffraction tomography was applied for visualizing individual cells and in vivo PHB granules and quantifying their physical properties such as weight, volume, density, and localization by measuring 3D refractive index distributions. Comparative analyses of native and recombinant PHB-producing strains revealed distinctive characteristics of PHB accumulation, providing insights into spatiotemporal localization of PHA granules in vivo. Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are intracellularly accumulated as distinct insoluble granules by various microorganisms. PHAs have attracted much attention as sustainable substitutes for petroleum-based plastics. However, the formation of PHA granules and their characteristics, such as localization, volume, weight, and density of granules, in an individual live bacterial cell are not well understood. Here, we report the results of three-dimensional (3D) quantitative label-free analysis of PHA granules in individual live bacterial cells through measuring the refractive index distributions by optical diffraction tomography (ODT). The formation and growth of PHA granules in the cells of Cupriavidus necator, the best-studied native PHA producer, and recombinant Escherichia coli harboring C. necator poly(3-hydroxybutyrate) (PHB) biosynthesis pathway are comparatively examined. Through the statistical ODT analyses of the bacterial cells, the distinctive characteristics for density and localization of PHB granules in vivo could be observed. The PHB granules in recombinant E. coli show higher density and localization polarity compared with those of C. necator, indicating that polymer chains are more densely packed and granules tend to be located at the cell poles, respectively. The cells were investigated in more detail through real-time 3D analyses, showing how differently PHA granules are processed in relation to the cell division process in native and nonnative PHA-producing strains. We also show that PHA granule–associated protein PhaM of C. necator plays a key role in making these differences between C. necator and recombinant E. coli strains. This study provides spatiotemporal insights into PHA accumulation inside the native and recombinant bacterial cells.