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

Abstract

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.

Long-term label-free assessments of individual bacteria using three-dimensional quantitative phase imaging and hydrogel-based immobilization

“…Traditional AST utilizes populational information of bacteria when measuring their susceptibility to antibiotics 45 . Our protocol can be implemented for not only population-based AST 24 , but also for assessing the drug susceptibility of individual bacterium, which has been indicated as a rapid alternative to the conventional time-consuming approaches 28 . A commercialized label-free individual bacteria AST method evaluates samples in an agarose-based environment using bright-field microscopy.…”

“…Bacteria can be used as cell factories for biopolymers, which are then used in medical and industrial applications 4 . By monitoring individual biopolymer production levels non-invasively, more efficient conditions for biopolymer production can be applied at medical and industrial levels 28 .…”

“…Bacteria are studied in more detail using three-dimensional (3D) QPI, which reconstructs the 3D refractive index (RI) tomogram from multiple 2D measurements of scattered light. 3D QPI facilitates single-cell level identification of bacteria 13 , studying the response of antimicrobial photodynamic therapy 27 , quantitative tracking of polymer synthesis in bacteria 28 , and time-lapse investigation of antibiotic-treated bacteria 25 .…”

Long-term label-free assessments of individual bacteria using three-dimensional quantitative phase imaging and hydrogel-based immobilization

“…Bacteria can be used as cell factories for biopolymers, which are then used in medical and industrial applications [4]. By monitoring individual biopolymer production levels non-invasively, more efficient conditions for biopolymer production can be applied at medical and industrial levels [28]. Moreover, our protocol can aid in cellular mechanism studies of individual bacteria.…”

“…Bacteria are studied in more detail using three-dimensional (3D) QPI, which reconstructs the 3D refractive index (RI) tomogram from multiple 2D measurements of scattered light. 3D QPI facilitates single-cell level identification of bacteria [13], studying the response of antimicrobial photodynamic therapy [27], quantitative tracking of polymer synthesis in bacteria [28], and time-lapse investigation of antibiotic-treated bacteria [26].…”