| Autor: |
Parrell D; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA.; Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726, USA., Olson J; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA., Lemke RA; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA.; Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726, USA., Donohue TJ; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA.; Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726, USA.; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA., Wright ER; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA.; Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726, USA.; Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Morgridge Institute for Research, Madison, WI 53715, USA. |
| Abstrakt: |
Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds. |
