Free PHB granules, i.e. PHB granules that were not in contact PX-478 mw to the nucleoid GSK3326595 region were not observed. Apparently, constitutive over-expression of phaM resulted in formation of an increased number of small and nucleoid-attached PHB granules. If PhaM is responsible
for the formation of small granules and for the close contact to the nucleoid region, deletion of phaM should have a phenotype. In fact, R. eutropha ∆phaM cells accumulated only very few (0–2) PHB granules that were significantly larger in diameter than those of the phaM over-expressing mutant or of the wild type (Figure 5). Since the diameters of PHB granules of the ∆phaM strain were considerably larger even at early time points a precise analysis whether or not the granules were attached to the nucleoid region was difficult. In most ∆phaM cells the PHB granules were still located close to the nucleoid; however, VX-809 manufacturer at least in some cells a detachment of PHB granules from the nucleoid region could not be excluded for the wild type or for the phaM over-expressing strain. A clear decision whether the absence of PhaM resulted in detachment from the nucleoid can, however, not be made. Since
R. eutropha expresses at least one other protein with DNA-binding and PHB-binding property (PhaR) [30, 31] it might be that PhaR also contributes to association of PHB with DNA. In summary, our data on mutants with altered expression of PhaM clearly show that number, diameter and subcellular localization of PHB granules depends on the presence and concentration of PhaM. Time course of formation and localization of PHB granules in R. eutropha over-expressing PhaP5 PhaP5 had previously been identified as a phasin in R. eutropha by its in vivo interaction with PhaP2 and other phasins . Remarkably, PhaP5 also interacted
with PhaM. To investigate the influence of PhaP5 on 5-Fluoracil PHB granule formation the phaP5 gene was cloned in a broad host range plasmid (pBBR1MCS-2) under control of the strong and constitutive phaC1 promotor (PphaC), transferred to R. eutropha H16 and HF39 via conjugation and investigated for PHB granules formation and localization under PHB permissive conditions (Figure 6). In case of strain HF39 a eypf-phaP5 fusion was cloned and used to confirm localization of PhaP5 on the PHB granules by fluorescence microscopy. Controls showed that free eYfp is a soluble protein in R. eutropha (Figure 7). Figure 7 Fluorescence microscopical (FM) investigation of R. eutropha H16 (pBBR1MCS-2-P phaC – eyfp -c1) with over-expression of eYfp (a); R. eutropha H16 (pBBR1MCS-2-P phaC – phaP5 ) with over-expression of PhaP5 (b), and R. eutropha H16 (pBBR1MCS-2-P phaC -eyfp- phaP5 ) with over-expression of eYfp-PhaP5 fusion (c) at various stages of PHB formation.