Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Abstract

Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homooligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wildtype cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His 6 and PhaM-His 6 showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed. Ralstonia eutropha is the model organism for research on microbial synthesis of short-chain-length polyhydroxyalkanoates (PHA SCL ) such as poly(3-hydroxybutyrate) (PHB) or copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHB/HV). PHB and PHB/HV are produced on an industrial scale of 10 4 to 10 5 tons per annum worldwide. For overviews on polyhydroxyalkanoate (PHA) metabolism, see references 1 to 8. Despite many years of intensive research on the molecular biology of PHA accumulation in Eubacteria and Archaea (9, 10), several aspects of PHA granule formation and subcellular localization of PHB granules have not been solved. Two models of PHB granule initiation, (i) the budding model (PHB granules initiate in the cytoplasm membrane and the growing PHB granule blebs out from the membrane at late stages) and (ii) the micelle model (PHB synthase PhaC1 is soluble in the cytoplasm and forms micelles with other PhaC1 molecules and with the growing [hydrophobic] PHB chain) were discussed in the past (11). Recently, association of PHB granules with the cytoplasm membrane could be excluded in R. eutropha by electron cryotomography (12). In our lab, we identified two new pro...

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

“…The resolution of the gel did not allow a precise determination of the apparent molecular mass. This result was in agreement with previous analysis that suggested the PhaM is present in oligomeric form, most likely as a dodecamer (43). Remarkably, also PhaC1 formed high-molecular-mass complexes of Ն300 kDa.…”

“…Nucleoid occlusion of PHB granules (50) in growing cells might be one explanation for the frequent observation of periphery-localized PHB granules, and this assumption would allow to keep the micelle model. However, the identification of PhaM as a novel PHB-and DNA-bound protein (27,29) and the in vivo interaction of PhaC1 and PhaM in R. eutropha (43) is in disagreement with the assumption of a random localization of PhaC1 in the cytoplasm. It is more compatible with a third model, the scaffold model, in which the PHB synthase PhaC1 is attached to an internal cellular structure, most likely to the bacterial nucleoid.…”

“…We showed by bimolecular fluorescence complementation that PhaM and PhaC1 were localized close to each other in vivo in R. eutropha (43). We therefore hypothesized that PhaM could also be important in vivo for PHB synthase activity.…”

“…Purified His 6 -PhaC1 was concentrated to 4 mg/ml via ultrafiltration and was then shock-frozen in liquid nitrogen and stored at Ϫ80°C until use. Purification of PhaM-His 6 and PhaP5-His 6 was performed as described previously (43).…”

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…However, Neumann et al proposed that PhaP Re occurs as a trimer (17), and some phasins have been reported to form other kinds of oligomers, such as dimers and dodecamers (corresponding to PhaP5 and PhaM from R. eutropha, respectively) (22). A common structural characteristic of phasins is the presence of coiled-coil interacting regions.…”