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1.
Figure 5

Figure 5. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Time course of PHB granule formation in R. eutropha H16 ∆phaM. All preparations were performed as described in legend to Figure 1. Note, deletion of phaM resulted in formation of decreased number of big PHB granules. Incubation times in NB-gluconate medium for 0 min (a), 30 min (b), 60 min (c) and 180 min in (d). Bar 0.2 μm.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
2.
Figure 4

Figure 4. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Individual cell of R. eutropha H16 with constitutive over-expression of PhaM after 1 h of PHB permissive conditions. Three invaginations of the cell wall (= 4 cells) are a visible indication that the last two cell-divisions have not been finished. All preparations were performed as described in legend to Figure 1. Note, presence of four individual, well-separated clusters of PHB granules apparently each bound to the nucleoid regions of the division-inhibited cell. Bar 0.5 μm.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
3.
Figure 2

Figure 2. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Time course of PHB granule formation in R. eutropha H16 and HF39. Images of both strains are shown alternately after different times of PHB permissive growth as indicated. All preparations were performed as described in legend to Figure 1. Note, increased number of PHB granules in strain H16 compared to strain HF39 at longer growth times. Strain HF39 [(a) 0 min after transfer to fresh NB-gluconate medium; (d), 10 min after transfer; (f) 40 min and (i) 3 hours)]. Strain H16 [(b) 0 min after transfer to fresh NB-gluconate medium; (c) 10 min; (e) 30 min; (g) 1 hour and (h) 3 hours]. Size of bar as indicated.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
4.
Figure 7

Figure 7. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Fluorescence microscopical (FM) investigation of R. eutropha H16 (pBBR1MCS-2-PphaC-eyfp-c1) with over-expression of eYfp (a); R. eutropha H16 (pBBR1MCS-2-PphaC-phaP5) with over-expression of PhaP5 (b), and R. eutropha H16 (pBBR1MCS-2-PphaC-eyfp-phaP5) with over-expression of eYfp-PhaP5 fusion (c) at various stages of PHB formation. PHB-free cells from 24 h old seed cultures on NB were transferred to fresh NB medium supplemented with 0.2% gluconate and grown at 30°C. FM-images of samples taken at time points as indicated were generated 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.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
5.
Figure 6

Figure 6. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Time course of PHB granule formation in R. eutropha with over-expression of phaP5. All preparations were performed as described in legend to Figure 1. Note, over-expression of phaP5 resulted in formation of two clusters of 2–5 individual PHB granules. Remarkably, most PHB granules were clearly detached from nucleoid region (arrowheads). Images were prepared from eYfp-PhaP5 over-expressing cells (except for (f) in which PhaM was over-expressed in strain H16) to directly compare with cells of Figure 7. No difference was detectable to R. eutropha H16 cells with over-expression of PhaP5. Incubation times in NB-gluconate medium for 0 min (a), 10 min (b), 20 min (c), 40 min (d), 90 min (e and f), 180 min (g). Bar 0.2 μm.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
6.
Figure 3

Figure 3. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

Time course of PHB granule formation in R. eutropha with over-expression of PhaM or eYfp-PhaM. All preparations were performed as described in legend to Figure. 1. Note, over-expression of PhaM resulted in formation of an increased number of small PHB granules. PHB granules generally were in close contact to nucleoid region. Strain H16 with over-expression of PhaM in (a, 0 min; c, 10 min; f, 40 min; h, 60 min; k, 240 min). Strain HF 39 (with over-expression of eYfp-PhaM) (b, 0 min; d, 10 min; e, 20 min; g, 40 min; i, 90 min; j, 180 min). Bar 0.2 μm.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.
7.
Figure 1

Figure 1. From: PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

TEM images of R. eutropha H16 (a) and of R. eutropha HF39 (b) after 24 h of growth on NB medium (=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.

Andreas Wahl, et al. BMC Microbiol. 2012;12:262-262.

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