New insights into PhaM-PhaC-mediated localization of polyhydroxybutyrate granules in Ralstonia eutropha H16

Abstract

The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaCC319A protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size.