Nanoscale Imaging and Proteomic Analysis Reveal a Tightly Regulated, Amorphous Granular Biopolymer Structure inside Bacteria.

Polyhydroxyalkanoates (PHAs) are biopolyesters that accumulate as cytosolic cell inclusions in many bacteria, enhancing resistance to environmental stresses. However, the structure, spatial localization, plasticization effects, and mechanical properties of PHA granules in vivo remain underexplored. Here, Cupriavidus necator H16 grown under nutrient-rich and limited conditions was investigated across a 72 hour accumulation cycle using orthogonal techniques including atomic force microscopy infrared (AFM-IR) spectroscopy, contact resonance mechanical analysis, optical photothermal infrared spectroscopy, nano-thermal analysis, and proteomics to map polyhydroxybutyrate (PHB) granule evolution. Across the PHB production cycle, the PHB content increased from 34 to 69% after 72 hours in minimal media, while in vivo crystallinity reached a maximum of 20% compared to 42-63% when extracted. Ester carbonyl band shifts (1720-1740 cm-1) indicated mostly amorphous PHB in vivo, while contact resonance mapping showed that the granules were mechanically softer than the surrounding cellular matrix. Notably, AFM-IR revealed membrane patterning consistent with spinodal decomposition, representing the first reported observation of spinodal-like membrane organization in C. necator. Proteomics identified increased production of PHB biosynthesis, regulation, and phasin proteins during the stationary phase, supporting phasin-assisted suppression of crystallization. Whole-pathway analysis further revealed stronger perturbations in formate oxidation and polyphosphate metabolism than in PHB biosynthesis alone, highlighting broader metabolic shifts. This study establishes a link between PHB accumulation, crystallinity, mechanical softness, and granule-associated protein production in C. necator, yielding a plasticized intracellular polymer state relevant to flexible bioplastic and biomedical material design.