PMID:28389545

Citation	Bresan, S and Jendrossek, D' (2017) New Insights in PhaM-PhaC-mediated Localization of PHB Granules in Ralstonia eutropha H16. Appl. Environ. Microbiol. '
Abstract	Formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha is controlled by PhaM that 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 to attach PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in a detachment of formed PHB granules from the nucleoid. Instead, a formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells was 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 co-localization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaC(C319A) proteins 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 at later stages from formed PHB granules resulting in an inhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB permissive conditions at a certain size.Importance PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells and for association of formed PHB granules to the nucleoid. Here we found out that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules to the nucleoid. Furthermore, we followed PHB granule formation by time lapse microscopy and provide evidence for an aging of PHB granules that is manifested by a detachment of previously PHB granule-associated PhaM and PHB synthase.
Links	PubMed Online version:10.1128/AEM.00505-17
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CUPNH:Q0KFC8		GO:0043590: bacterial nucleoid	ECO:0000314:		C		Determination of effects, c-terminal domain knock out from PHaM (Hypothetical membrane associated protein), had lead to a change in localization, fusion with the gene for the enhanced yellow fluorescent protein (eyfp). Constitutively expressed PhaMWT 182 -eYFP showed a strong 183 fluorescence in the nucleoid region of R. eutropha (Ralstonia eutropha) cells. Fig. 2A Left column Cupriavidus necator, phaM/H16_A0141	complete CACAO 12579
CUPNH:Q0KFC8	Colocalizes with	GO:0060187: cell pole	ECO:0000315:		C		Mutant type, PhaM∆C 191 -eYFP(Hypothetical membrane associated protein), had knocked out C-terminal domain of PhaM, which changed localization to cell poles, and areas of future septum formation when viewed from results by Imaging in bright field and/or staining with Nile red. Also showed co- localization of PhaM∆C 191 -eYFP with formed PHB granules in Ralstonia eutropha organism. Fig 2A right column.	complete CACAO 12582

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