Studies on the mechanism of PHB granule formation in Ralstonia eutropha

Abstract

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Polyhydroxybutyrates (PHBs) are biodegradable polymers produced by bacteria in the form of insoluble granules for carbon storage. Insights into the mechanisms of granule formation are important for improving commercial PHB production and engineering new alternatives to petroleum-based plastics. Using both biochemical and imaging methods, we examined PHB granule formation in the model gram-negative bacterium, Ralstonia eutropha H16. In order to examine initiation and elongation of PHB polymerization in vivo, an N-terminally Strep2-tagged PHB synthase was isolated from R. eutropha using a strain in which the native phaC gene was replaced with the affinity tagged gene. Activity assays of the Strep2-PhaCRe revealed no lag phase in CoA release, indicating the presence of PHB primed synthase in vivo. Further analysis showed that the primed synthase was associated with soluble, large molecular weight PHB and the phasin protein PhaP1 in a high molecular weight (HMW) complex. Based on the composition and activity of the HMW complex, we propose that we have isolated soluble granule precursors, supporting a cytoplasmic model for granule formation. The stoichiometric relationships between PhaC, PhaPI, and PHB in vivo were next examined in wt R. eutropha as a function growth time in nitrogen limited PHBHigh medium. Studies demonstrated that the ratio of PHB/PhaP1 remained constant throughout growth, between 1 and 2, while the PHB/PhaC ratio increased with time, suggesting increasing re-initiation events. These relationships were further measured in PhaP1 deletion and overexpression strains. The structural and kinetic effects of PhaP1 on in vitro reactions with PhaC and [1- C]-HBCoA were also examined. The results suggest a model for granule self-assembly by PhaC, PhaP 1, and HBCoA alone, in which PhaP 1 helps to "fold" the PHB chains and the PhaP1/PhaC ratio controls the degree of polymer reinitiation. Finally, PHB granule localization during formation was examined in live R. eutropha cells using PhaP1 labeled with the biarsenical fluorescent dye FlAsH. Fluorescence microscopy showed no specific polar or membrane localization during granule formation in PHBHigh medium. In collaboration with the Jensen lab at Caltech, granule localization was also studied using cryoelectron tomography. Together, the imaging results eliminate a membrane budding model for granule formation.by Mimi Cho.Ph.D