Scientists have reported the first-ever time-lapse movie of polyhydroxyalkanoate granule formation in bacterial cells, made possible with the help of 3D holographic microscopy.
Amid the growing plastic waste crisis, metabolic engineering researchers from the Korea Advanced Institute of Science and Technology (KAIST) have successfully observed how bioplastic granules of polyhydroxyalkanoates (PHAs) accumulate in living bacterial cells with the help of 3D holographic microscopy, bringing new knowledge into the biosynthesis of sustainable plastics.
Widely endorsed as an eco-friendly alternative to synthetic plastics, PHAs are bacterial polyesters used to create biodegradable plastics. But before they became the building blocks of bioplastics, they were originally energy and carbon storage materials, which existed as insoluble granules and synthesised as a result of bacteria growing in an environment with excess carbon sources. And up until recently, the exact mechanisms behind the synthesis of these granules have remained elusive.
Many researchers have attempted to analyse these PHA granules in vivo using a range of tools such as fluorescence microscopy, transmission electron microscopy, and electron cryotomography. However, transmission electron microscopy relies on the statistical analysis of multiple 2D snapshots of fixed cells or the short-term monitoring of the cells, and fluorescence techniques make use of fluorescence labelling or dye-staining that can also affect the viability of cells. Therefore, scientists could not examine the real-time accumulation of PHAs in live cells. Due to these technical limitations, scientists have long struggled to fully understand the underlying processes of PHA granule formation.
Only now were researchers finally able to gain a full view of PHA granule formation and distribution in live bacterial cells. With their newly developed 3D holographic microscopy, Distinguished Professor Sang Yup Lee, Physics Professor YongKeun Park, and colleagues were able to conduct a 3D quantitative label-free analysis of PHA granules in individual live bacterial cells.
Using optical diffraction tomography, the team measured the refractive index distributions of bacterial cells and reconstructed a 3D visualisation of the cells. To study PHA accumulation, they chose to examine Cupriavidus necator, the most-studied natural PHA producer, and a recombinant Escherichia coli, which was transformed to express Cupriavidus necator’s PHB (a form of PHA) biosynthesis pathway. Their efforts were successful as they were able to observe the formation and growth of PHA granules in both cells, and even recorded the processes in time-lapse movies.
Besides observing the formation of PHAs, the team also performed a quantitative analysis of the cells and intracellular PHA granules at a single-cell level. After analysing hundreds of single cells accumulating PHA granules, the team detected important features concerning the density and localisation of PHA granules. Comparisons between C. necator and E. coli revealed a key protein PhaM that allows the characteristics of PHA granules in the recombinant E. coli to be more similar to those of C. necator.
As the first study ever to report 3D time-lapse clips demonstrating the processes of PHA granule formation, cell growth, and cell division of bacterial cells in their native state, the findings of their study have shed valuable insights on biosynthesising sustainable substitutes for petroleum-based plastics.
“This study provides insights into the morphological and physical characteristics of in vivo PHA as well as the unique mechanisms of PHA granule formation that undergo the phase transition from soluble monomers into the insoluble polymer, followed by granule formation. Through this study, a deeper understanding of PHA granule formation within the bacterial cells is now possible, which has great significance in that a convergence study of biology and physics was achieved. This study will help develop various bioplastics production processes in the future,” commented Professor Lee.
Source: Choi et al. (2021). Three-dimensional label-free visualization and quantification of polyhydroxyalkanoates in individual bacterial cell in its native state. Proceedings of the National Academy of Sciences, 118(31).