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.

聚羟基链烷酸酯 (PHA) 是可生物降解的聚酯，可通过各种微生物在细胞内以不同的不溶性颗粒形式积累。PHA 作为石油基塑料的可持续替代品引起了广泛关注。然而，在单个活细菌细胞中 PHA 颗粒的形成及其特征，例如颗粒的定位、体积、重量和密度，尚不清楚。在这里，我们通过光学衍射断层扫描 (ODT) 测量折射率分布，报告了对单个活细菌细胞中 PHA 颗粒进行三维 (3D) 定量无标记分析的结果。Cupriavidus necator细胞中 PHA 颗粒的形成和生长，这是研究得最好的天然 PHA 生产者和重组体对含有C. necator poly(3-hydroxybutyrate) (PHB) 生物合成途径的大肠杆菌进行了比较检查。通过对细菌细胞的统计 ODT 分析，可以观察到 PHB 颗粒在体内的密度和定位的独特特征。与C. necator相比，重组大肠杆菌中的 PHB 颗粒显示出更高的密度和定位极性，表明聚合物链更密集，颗粒倾向于分别位于细胞极。通过实时 3D 分析对细胞进行了更详细的研究，显示了在天然和非天然 PHA 生产菌株中，PHA 颗粒的处理与细胞分裂过程有何不同。我们还表明，C. necator 的 PHA 颗粒相关蛋白 PhaM在造成C. necator和重组大肠杆菌菌株之间的这些差异中起关键作用。这项研究提供了对天然和重组细菌细胞内 PHA 积累的时空见解。