2% gluconate and grown at 30°C. FM-images of samples taken at time points as indicated were generated Semaxanib nmr after

staining with Nile red in red channel (top rows) or without staining in green channel (bottom rows) or. Note, individual PHB granules of PhaP5 or eYfp-PhaP5-expressing cells near cell poles or at mid cell were not resolved in FM images as in TEM images (Figure 6). Bar 3 μm. As in the case of PhaM, no difference in number, size and localization of PHB granules was observed for the over-expressed eYfp-PhaP5 fusion in comparison to over-expression of PhaP5 alone. Growth and accumulation of PHB were similar in the recombinant strains as in the wild type. However, when the time-course of PHB granule formation and localization was investigated by TEM-analysis Mizoribine manufacturer remarkable differences to the wild type were observed for the PhaP5 over-expressing strains (Figure 6): PHB granules were formed in aggregated clusters of in average 2–6 granules in most cells near both cell poles of the rod-shaped cells. These clusters could not be resolved

by FM-analysis (Nile red staining) and resulted in the impression of only two (large) PHB granules near the cell poles (Figure 7). The number of individual granules visible in TEM images was increased but the diameter was decreased compared to wild type granules. In most cells, the PHB granule clusters or at least individual PHB granules of a cluster were clearly detached from the nucleoid region (see arrowheads in Figure 6). In conclusion, over-expression of PhaP5 has an impact on number, size and localization

of PHB granules and leads to detachment of the granules from the nucleoid. This can be explained by binding of over-expressed PhaP5 to PhaM molecules thus preventing PhaM from binding to DNA and/or to Edoxaban PhaC. Alternatively, a competitive displacement of PhaM molecules from PHB granules surface by over-expressed PhaP5 could be responsible for the phenotype. Number and localization of PHB granules in a ∆phaP5 strain were, however, not significantly changed in comparison to wild type (data not shown). Conclusions Our data clearly show that formation and localization of PHB granules occurs not randomly but is specifically controlled in R. eutropha. Other examples of species with non-random localization of PHB granules are Rhodospirillum rubrum[33], Haloquadrata walsbyi, Azotobacter vinelandii, Beijerinckia indica[34], Caryophanon latum[35] and Hyphomicrobium SIS3 facile (supplementary material of [32]). However, we do not know whether attachment of PHB or PHA granules to the DNA is a general feature of PHB or PHA accumulating bacteria. PHB granules in R. eutropha are attached to the nucleoid via PhaM. Our conclusion is supported by previous TEM analysis of others if the “dark-stained mediation elements” are interpreted as denatured chromosomal DNA [36, 37].