For this, the culture was transferred to Falcon tubes and immediately cooled on ice. Cells were centrifuged (4°C) and washed with 1 mL of ice-cold PBS (phosphate-buffered saline consisting of 50 mM potassium phosphate and 0.8% NaCl, pH 7.2). Cells were resuspended with 0.8 mL PBS and solutions of formaldehyde

(final concentration 0.3 to 1.0%) and glutardialdehyde (0.2 to 1.0%) were added for fixation. Samples were stored on ice overnight. Table 1 Strains and plasmids used in this study Strain Relevant characteristic Source or reference GDC 0449 Escherichia coli JM109 Cloning strain   E. coli S17-1 Conjugation strain [45] Ralstonia eutropha H16 Wild type strain, PHB accumulation DSMZ 428 Ralstonia eutropha HF39 Streptomycin resistant derivate of H16 [22, 39] R. eutropha H16 ∆phaP5 Chromosomal deletion of phaP5 [22] R. eutropha H16 ∆phaM Chromosomal deletion of phaM [32] Plasmid Relevant feature(s) Source or reference pBBR1MCS-2 broad host range vector [46] pBBR1MCS2- PphaC-eyfp-c1

Constitutive eYfp over-expression [22] pBBR1MCS-2- P phaC -eyfp-phaP5 Fusion of PhaP5 to C-terminus of eYfp [22] pBBR1MCS-2- P phaC –eyfp-phaM Fusion of eYfp to N-terminus to PhaM [32] pBBR1MCS-2- P phaC –phaP5 Constitutive over-expression of PhaP5 this study pBBR1MCS-2- P phaC –phaM Constitutive over-expression of PhaM this study Preparation of cells for TEM analysis Fixed cells were washed three times with 1 mL PBS+10 mM glycine to remove excess of aldehydes. An aliquot of the cells was taken for fluorescence microscopy. The cell pellet of the third washing step was resuspended AZD2014 in vitro with PBS in a final volume of 100 μL. Cells were added to an equal volume of a 2% (in PBS) agar solution (prewarmed to 50°C in a 2 mL Eppendorf tube using prewarmed pipette tips), mixed and centrifuged for ≈ 10 s at room temperature to obtain a high cell concentration at the bottom of the agar. The agar was cooled on ice. The agar block containing fixed R. eutropha cells was removed from the Eppendorf cups using a steam of nitrogen gas applied with a

capillare to the bottom of the Eppendorf tube and was cut into more or less cube-shaped pieces (≈ 1 mm3). The cells were dehydrated by incubation of the agar cubes in a series of subsequent dehydration steps using: Sclareol 15% methanol on ice for 15 min, 30% ethanol for 30 min on ice, and subsequent 30 min incubation steps at – 20°C using 50%, 70%, 96% and 100% (twice) ethanol. Subsequently, the dehydrated cubes were transferred to a solution consisting of ethanol and LR white resin (3:1) and incubated at room temperature for 2 h before the solution was exchanged against pure LR white and incubated at 4°C for at least 2 h (or overnight). Several cubes were then transferred to gelatine capsules, filled with LR white and polymerized at 50°C (or 60°C) for 30 h (or 24 h). The solidified samples were stored in the dark at room temperature until use.