When cells were investigated that had been grown for >1 h permissive for PHB synthesis the number and size of the granules further increased. Strain H16 accumulated in average more granules (up to 12) than strain HF39 (1 to 4). Since the diameter of accumulated PHB granules increased by time the volume of the granules also increased and the association of the granules with the nucleoid became less obvious and could not be differentiated from nucleoid exclusion; however PND-1186 it should be noted that for all cells shown in Figure 2, in which PHB granules and the nucleoid were visible, an association of the granules with the nucleoid is evident. In conclusion, the data suggest that PHB granules are rapidly formed under
permissive conditions (within 10 min) and apparently are attached to the nucleoid region. Since PhaM binds to both DNA and PHB we speculated that PhaM is responsible for the association of PHB granules with the nucleoid (see below). Time course of formation and subcellular localization of PHB granules in R. eutropha that over-express PhaM PhaM represents a new type of PHB granule associated protein and has multiple functions. It had MK-8931 cost been identified by its in vivo interaction with PHB
synthase PhaC1 in a two-hybrid screening assay . FM analysis revealed that PhaM is not only able to bind to PHB granules but also to the nucleoid region in R. eutropha. Moreover, purified PhaM was able to bind to genomic DNA in vitro as indicated in gel mobility shift experiments. To investigate the effect of PhaM on PHB granule formation the phaM gene was over-expressed constitutively from the phaC1 promotor. Figure 3 shows the time course of PHB granule formation in the PhaM-over-expressing transconjugant of R. eutropha H16 and HF39. No difference in number, size or localization of PHB granules was found when PhaM-over-expressing cells were compared with eYfp-PhaM over-expressing cells and MLN2238 ic50 confirmed that the presence of an eYfp tag did not change subcellular localization very of fusion proteins. Most cells were free of PHB granules at zero time and the
nucleoid region could be differentiated from the cytoplasm by the different degree of adsorbed staining material similar to wild type cells. PHB granules appeared already after 10–20 min of incubation under PHB permissive conditions. At later time points the number of PHB granules strongly increased up to several dozens. The granules were considerably smaller in diameter (< 100 nm) compared to wild type cells at all stages of growth and the granule size increased only little after longer incubation times at PHB permissive conditions. Remarkably, the granules were not randomly distributed in the cells but were exclusively in contact with or in close neighbourhood to the nucleoid. The PHB granules covered the complete surface of the nucleoid region in some cells. Occasionally, long cells were observed that apparently were inhibited in cell division (Figure 4, 3 h).