Results and discussion Time course of PHB granule formation in R. eutropha HF39 and H16 To study the formation and localization of PHB granules in R. eutropha we used R. eutropha MM-102 in vivo strains H16 and HF39. Both strains have wild type properties with respect to PHB metabolism Pictilisib research buy and easily form PHB granules during growth on rich media such as NB medium media. Strain HF39 is a spontaneous streptomycin resistant mutant of strain H16 and has often been used in place of strain H16 in conjugation experiments because of simplified counter selection of the donor [39]. In this study, the same results were obtained for both strains with the exception that strain HF39 grew slightly slower and produced in average

a lower number of PHB granules

than strain H16. Although R. eutropha strains H16 and HF39 intermediately accumulated PHB during growth on NB-medium more than 95% of the cells were free of PHB granules in the stationary growth phase after 24 h. Cells that still had PHB granules after this time period (<5%) often were division-inhibited (cells > 10 μm in length) and LY2874455 manufacturer many of them were dead as revealed by staining with propidium iodide (images not shown). In conclusion, most living cells of the late stationary growth phase of R. eutropha on NB-medium were free of accumulated PHB. To monitor the time course of PHB granule formation we transferred PHB-free stationary R. eutropha cells to fresh NB-medium that had been additionally supplemented with 0.2% sodium gluconate. This increased the C to N ratio of the medium and promoted PHB accumulation. Samples were taken at zero time and after 10 min to several hours

of growth. Harvested cells were chemically fixed, embedded in a low viscosity acrylic resin and subjected to thin section electron transmission microscopy. PHB granules poorly bind heavy atom stains and therefore have an electron-transparent (“white”) appearance. The results are as shown in Figures 1, 2, 3, 4, 5 and 6. Figure 1 TEM images of R. eutropha H16 (a) and of R. eutropha HF39 (b) after 24 h of growth on NB medium Tideglusib (=zero control [t=0 min after transfer to fresh NB-gluconate medium]). Cells were harvested, fixed and prepared for TEM as described in method section. All thin sections were stained with uranyl-acetate and lead citrate. Arrowheads indicate condensed cytoplasm resulting in an electron-transparent fringe between cytoplasm membrane and cytoplasm. Short arrows indicate the border between cytoplasm and denatured nucleoid. The long arrow in the left cell of (a) points to a small globular structure most likely representing an electron-transparent (“white”) remaining, not completely mobilised PHB granule. Note, the PHB granule is in close contact to nucleoid region. Bar represents 0.2 μm. Figure 2 Time course of PHB granule formation in R. eutropha H16 and HF39.