Quantitating Storage Granule Size, Accumulation, and Localization in Rhodobacter sphaeroides Using Cryo-Electron Tomography and Light Microscopy

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Bacteria employ many mechanisms for resisting and responding to environmental stressors. In this work we explore the production of polyhydroxybutyrate (PHB) and polyphosphate (PP) granules in response to antibiotic stress from chloramphenicol. Storage granules in bacteria and other organisms are produced to generate a cell structure highly concentrated in building blocks used for cellular metabolism. In the case of PHB, excess carbon in the form of acetate is shuttled to storage for later use. For PP, excess energy in the form of ATP is catalyzed into PP by the polymerization of inorganic phosphate. Storage granules such as these offer a unique structure that may prove valuable as a primary resource in the production of biofuels, bioplastics, and other biologically derived products. These structures were characterized in the facultative alpha-proteobacterium Rhodobacter sphaeroides using cryo-electron tomography (cryo-ET), fluorescence light microscopy, and biochemical purification. This work will advance our understanding of the production and localization of these structures in a model organism. R. sphaeroides cultures were grown at 30°C and 200 RPM shaking in Sistrom’s minimal medium until the culture reached an OD600 of 0.4. Cells were then treated with the translation blocking antibiotic chloramphenicol at 200 μg/mL concentration for 6 hrs. Suspensions of the cells were stained with the membrane dye FM4-64 and deposited onto 200 mesh R2/1 copper Quantifoil grids in 5 μL aliquots, blotted, and plunge frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific). An additional sample was used for epifluorescence microscopy. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) data were collected using a Titan Krios G3i TEM (ThermoFisher Scientific) operated at 300 kV, equipped with a Bioquantum energy filter and a K3 direct electron detector (Gatan). Single axis tilt series were acquired using SerialEM [1], with an increment of 2° covering −60° to +60°, or an increment of 3° covering −60° to +60° and a cumulative dose under 45 e-/Å2 at a defocus range between −4 and −10 μm. Tomograms were reconstructed using IMOD/eTomo [2], and 3D rendering was performed using EMAN2 neural network segmentation training [3]. We observed the production and localization of PHB and PP in the bacterium R. sphaeroides with and without treatment with chloramphenicol (Cm). Cryo-ET imaging showed that PHB granules grow significantly upon Cm treatment. High throughput segmentation and 3D model analysis of the granules illustrated that the volume of PHB expands by nearly an order of magnitude within the cell. To corroborate these observations, the amount of PHB on the per cell basis was measured by gas chromatography mass spectrometry, after cell digestion and PHB extraction. These cell extract results revealed a similar level of PHB accumulation when compared to quantifications from the cryo-ET data. We also demonstrated that PP granules grow significantly in size upon Cm treatment, although not to the scale of PHB accumulation. Using fluorescence microscopy, we showed that GFP-tagged PHB granules are generally polarly localized in cells at all stages of the cell cycle, and in untreated cells the granules are localized at the old pole of resulting daughter cells. Upon Cm treatment, PHB granules maintain polar localization. Interestingly, a mid-cell localized PHB granule was also seen in cells in late stages of cell division. It is presumed that this additional localization promotes the effective partitioning of the additional PHB biomass upon Cm treatment. Finally, it was observed that PHB and PP granules commonly colocalize. Our data demonstrate that antibiotic stress from Cm treatment significantly impacts the size, accumulation, and localization of granule-based storage structures in R. sphaeroides. A block in translation by chloramphenicol, resulting in cells that can no longer synthesize new proteins. This may lead to cells stockpiling nutrients and energy in the form of PHB and PP granules as mechanisms to resist or mitigate the antibiotic stressor. Accumulation of these structures is of interest for potential bioproduct related research, especially in the case of PHB, which has been considered for bioplastics research [4]. By completing this work, we aim to better understand bacterial stress response mechanisms, and advance the ability to accumulate valuable biological materials for bioproducts research [5]. PHB and PP accumulate in granules upon Cm treatment. An R. sphaeroides culture was grown in SIS medium and left to grow (A) or treated with 200 μg/mL Cm (B). Chloramphenicol treatments increase PHB (red arrows) accumulation and granule size. A.) WT R. sphaeroides cells were observed to have ∼6-7 PHB granules (red arrows) per cell. B.) Following 200 μg/mL chloramphenicol treatments the number of PHB granules was less (∼2-3 per cell) and the amount of PHB per cell had increased significantly. Scale bars are 500 nm. Segmentation of Rhodobacter sphaeroides cells. A. A central slice through a cryo-tomogram of an untreated cell. B. A 3D segmentation model of the same cell depicting the outer membrane (green), inner membrane (blue), PP (purple), and PHB (cyan). Scale bars are 500 nm.

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