Formation of an organic-inorganic biopolymer: polyhydroxybutyrate

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Formation of an organic-inorganic biopolymer: polyhydroxybutyrate-polyphosphate Jennie C Hildenbrand, Simone Reinhardt, and Dieter Jendrossek Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Formation of an organic-inorganic biopolymer: polyhydroxybutyrate-polyphosphate Jennie C. Hildenbrand, Simone Reinhardt, Dieter Jendrossek* Institute of Microbiology, University of Stuttgart, Stuttgart, Germany

ABSTRACT: A considerable variety of different biopolymers is formed by the entirety of organisms present on earth. Most of these compounds are organic polymers such as polysaccharides,

polyamino

acids,

polynucleotides,

polyisoprenes

or

polyhydroxyalkanoates (PHAs), but some biopolymers can consist of solely inorganic monomers such as phosphate in polyphosphates (polyPs). In this contribution, we describe the formation of an organic-inorganic block-copolymer consisting of poly(3hydroxybutyrate) (PHB) and polyP. This was achieved by the expression of a fusion of the polyP kinase gene (ppk2c) with the PHB synthase gene (phaC) of Ralstonia eutropha in a polyP-free and PHB-free mutant background of R. eutropha. The fusion protein catalyzed both the formation of polyP by its polyP kinase domain and the formation of PHB by its PHB synthase domain. It was also possible to synthesize the polyP-PHB polymer in vitro with purified Ppk2c-PhaC, if the monomers, adenosine triphosphate (ATP) and 3-hydroxybutyryl-CoA (3HB-CoA), were provided. Most likely, the formed

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block copolymer (polyP-protein-PHB) turns into a blend of polyP and PHB after release from the enzyme.

KEYWORDS: Ralstonia eutropha, biopolymer, polyphosphate kinase, polyhydroxybutyrate synthase

INTRODUCTION Living cells can be considered as globular structures surrounded by membrane phospholipids that consist mainly of insoluble or water-solubilized, water-emulsified or water-suspended polymer mixtures. Most of these biopolymers are organic polymers such as polysaccharides (e. g. cell walls), polyaminoacids (proteins), polynucleotides (nucleic acids), polyisoprenes (rubbers in plants) and polyhydroxyalkanoates (PHAs in prokaryotes). However, one inorganic polymer, polyphosphate (polyP), is also present in species of all kingdoms of live and presumably is present in all organisms on earth1,2. PolyP is a reservoir for phosphorous and cations (Ca2+, Mg2+), and can also be considered as an energy reservoir as it can serve as phosphate donor for the biosynthesis of ATP or GTP in the reverse polyP kinase reaction. A second function of polyP is to substitute ATP in some kinase reactions (e. g. polyP-dependent NAD kinase3, polyP-dependent glucokinase4). Moreover, polyP has several additional functions: in microorganisms, it is involved in stress-resistance against reactive (oxidizing) compounds and in the pathogenicity of pathogenic bacteria. The biopolymer


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can also help to keep proteins in their native (active) states (chaperon function of polyP)5,6 7. In humans, polyP is part of the blood coagulation system (activation of Hageman factor [factor XII])8. PolyP in prokaryotes is synthesized by the action of polyP kinases from ATP (or GTP). Two major types of polyP kinases are currently known. One is polyP kinase 1 (PPK1) that has first been described in E. coli9 and consists of four domains (for details see10). PPK1s have molecular masses of ≈80 kDa and ppk1 homologs have been identified in many genome-sequenced prokaryotes. The other PPK type is PPK2, which is characterized by a PPK2-domain and has roughly half of the molecular mass of PPK1s (≈40 kDa). Three subtypes of PPK2s are currently known11. Prokaryotes have either PPK1 or PPK2 or can have both types of PPKs. Some bacterial species can have multiple ppk genes in the genome. Ralstonia eutropha, a widely used Gram-negative betaproteobacterium, is remarkable because it has seven PPKs: two of the PPK1 type (Ppk1a, Ppk1b) and five of the PPK2 type (Ppk2a, Ppk2b, Ppk2c, Ppk2d and Ppk2e)12. Poly(3-hydroxybutyrate (PHB) is a storage compound for carbon and energy and is synthesized in many prokaryotes during imbalanced growth when an excess of a suitable carbon source is available but growth is limited by the absence of another essential nutrient such as a nitrogen or phosphorous source13. Under such PHBpermissive conditions, many prokaryotic species form up to a dozen of PHB granules with 200 to 500 nm in diameter. PHB granules are highly organized supra-molecular


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complexes (carbonosomes) consisting of a polymer core and a surface layer of up to 14 different proteins in case of the model organism for PHB granule formation R. eutropha H1614-17. The key enzyme of PHB biosynthesis is the PHB synthase that catalyzes the polymerization of the monomeric precursor 3-hydroxybutyryl-coenzyme A (3HB-CoA) to PHB18,19. Recently, the 3-D structure of the C-terminal catalytic domain of PhaC of R. eutropha was solved20,21. The PHB synthase PhaC (≈64 kDa) of R. eutropha can be modified on the gene level by fusion of domains from other proteins to various parts of the PhaC protein without losing the PHB polymerizing function22. Several examples of a surface display of protein domains or of full proteins on PHB granules were described by the construction of gene fusion of phaC with foreign genes and expression in recombinant E. coli strains23,24. Inspired by these successful examples, we assumed that it should be possible to construct an active fusion protein consisting of the PHB synthase PhaC and another protein with a polymerase function. As a proof of principle we chose the polyP kinase Ppk2c of R. eutropha12 and fused the ppk2c gene to the phaC gene and expressed the ppk2c-phaC fusion in a PHB- and polyP-free background of R. eutropha. The formation of PHB and polyP granules was restored and chains of PHB granules and polyP granules arranged alternately were formed both in vivo and in vitro. MATERIALS AND METHODS Bacterial strains, plasmids and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli JM109 and S17 were used for


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cloning and conjugation procedures, respectively. Genes were expressed in E. coli BL21 (DE3)/pLys. All E. coli strains were grown on lysogeny broth (LB) medium supplemented with the appropriate antibiotics (depending on the strains and plasmids) at 37°C or as indicated. R. eutropha strains were grown on nutrient broth (NB, 0.8%, wt/vol) medium, with or without sodium-gluconate (0.2%, wt/vol) and 0.2% arabinose (wt/vol) as indicated, at 30°C.

Table 1: List of strains, primers and plasmids used in this study Strains

Relevant characteristic

Source/reference

Escherichia coli JM109 Cloning strain

DSMZ 3423

E. coli BL21 (DE3) Expression strain, Kmr pLysS

Novagen

E. coli S17-1

25

Conjugation strain

Ralstonia H16

eutropha wild type strain, source of phaC and DSMZ 428 ppk2c

Ralstonia ∆ppk-all

eutropha Chromosomal deletion of all seven known ppk genes

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Ralstonia ∆phaC

eutropha Chromosomal deletion of phaC gene

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Ralstonia eutropha Chromosomal deletion of phaC and of all ∆ppk-all, ∆phaC seven known ppk genes

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Primers pBBR_ppk_f

ggcagagagacaatcaacatATGACAGATCG CGACACCATCCAATCCTCC

pBBR_phaC_r

ggatcccccgggaattccatTCATGCCTTGGC TTTGACGTATCG


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pET_ppk_f

gggaattccatATGACAGATCGCGACAC CATCC

pET_phaC_r

gcgcctcgagTCATGCCTTGGCTTTGAC GTATCGC

Plasmids pET28a

His tag expression vector, Kmr

pET28a::ppk2c

Production of His6-Ppk2c

pET28a::phaC

Production of His6-PhaC

pET28a::ppk2c-phaC

Production of His6-Ppk2c-PhaC

this study

pBBR1MCS2

Broad-host-range vector, Kmr

29

pBBR1MCS2-PphaCeyfp-c1

Universal vector for construction of 3´fusion of eyfp under control of the PphaC promoter

30

pBBR1MCS2-PphaCphaC-eyfp

Broad-host-range vector with 3´-fusion of This study phaC to eyfp

pBBR1MCS2-PBAD

Broad-host-range vector with arabinose inducible promoter

pBBR1MCS2-PBADppk2c-phaC

Broad-host-range vector with ppk2c and This study phaC fusion under control of an arabinose inducible promoter

pCM62

Broad-host-range vector, Tcr

pCM62-PphaCmCherry-c1

Universal vector for construction of This study fusions to mCherry under control of the PphaC promoter

pCM62-PPhaC-ppk2cmCherry

Broad-host-range vector for expression of This study fusion of ppk2c to mCherry

Novagen this study this study

an Daniel Pfeiffer

31

Kmr = resistance to kanamycin; Tcr = resistance to tetracycline

Construction of a ppk2c-phaC fusion. Molecular cloning of the ppk2c-phaC fusion gene was done by Gibson Assembly31. The gene product was amplified by PCR with the ACS Paragon Plus Environment

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primers pBBR_ppk_f and pBBR_PhaC_r for the pBBR1MCS2-PBAD-ppk2c-phaC construct and pET_ppk_f, as well as pET_PhaC_r, for the pET28a-ppk2c-phaC construct. Cloning of the ppk2c-phaC fusion into the pBBR1MCS2-PBAD and pET28a was done with NdeI and XbaI or with NdeI and XhoI as restriction sites, respectively. Both constructs were cloned into E. coli JM109 and verified by PCR amplification and sequencing, before pET28appk2c-phaC was transformed into the expression strain E. coli BL21(DE3) pLysS. The pBBR1MCS2-PBAD-ppk2c-phaC construct was transformed into E. coli S17-1 and transferred to R. eutropha ∆ppk-all, ∆phaC by conjugation. Overexpression and protein purification. Overexpression of his6-phaC, his6-ppk2c and his6-ppk2c-phaC was performed in recombinant E. coli BL21(DE3) pLysS by induction with 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at an optical density at 600 nm (OD600) of 0.6 and subsequent incubation overnight at 30°C for pET28a::ppk2c, at 26°C for pET28a::phaC and at 20°C for pET28a::ppk2c-phaC. Cells were harvested by centrifugation at 4,000 g for 20 min at 4°C and disrupted with a French press (2 passages). After ultracentrifugation for 1 h at 100,000 g, His6-PhaC was purified in the presence of 0.05% (wt/vol) Hecameg by metal chelate affinity chromatography using Ni-agarose according to a protocol described in detail previously32. For purification of His6-Ppk2c and His6-Ppk2c-PhaC a different buffer was used (50 mM Tris/HCl, pH 8.0, 500 mM NaCl, 20% glycerol). Purified proteins were concentrated via ultrafiltration with Biomax® ultrafiltration filter discs (10 kDa cut-off) and then shock-frozen in liquid nitrogen to be stored at -80°C until use. ACS Paragon Plus Environment

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PHB synthase and polyP kinase assay. PHB synthase activity was determined by the detection of released coenzyme-A (CoA) from the substrate 3HB-CoA with Ellman’s reagent (dithionitrobenzoate, DTNB) at 412 nm. Additionally, fluorescence microscopy was used to visualize the synthesized PHB granules after staining with Nile red as described previously32,33. PolyP kinase activity determination was performed by following the formation of polyP granules microscopically after staining of formed polyP with 4,6-diamidin-2-phenylindol (DAPI, 100 µg/ml) and detection at a DAPIpolyP specific wavelength (see12 for more details). To this end, purified Ppk2c-PhaC protein (150 to 200 nM) was incubated in a total volume of 20 µl buffer (50 mM TrisHCl, pH 8 containing 20% (v/v) glycerol and 0.5 M NaCl) in the presence of either 2-5 mM 3HB-CoA or 5-20 mM ATP (or in the presence of both substrates) for 60 min at 30°C. In case that PHB synthase activity only was tested, the assay buffer consisted of 150 mM potassium phosphate buffer, pH 7.1, containing 0.2% glycerol and the 3HBCoA concentration was reduced to 0.5 mM. Enzymatically produced polyP was also detected by toluidine blue staining after electrophoretic separation in a polyacryl amide gel according to Losito et al.34. Quantification of PHB and polyP was done by conversion of PHB to 3-hydroxybutryrate methylester and subsequent gas chromatography35 and by conversion of polyP to monomeric phosphate (Pi) with exopolyphosphatase and detection of released phosphate with the malachite green assay36.


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Microscopical methods. Formation of PHB and polyP was followed by fluorescence microscopy using Nile red (10 µg/ml DMSO, Nile red solution at 1 µg/ml) and DAPI (0.1 mg/ml in distilled H2O). Images were taken with a digital camera (Hamamatsu Orca Flash 4.0 sCMOS camera) and processed with Nikon imaging software. PHB granules could also be visualized by bright field microscopy. To image in vitro prepared PHB or polyP polymers, 8 µl portions of the stained samples were immobilized on agarose pads (1% wt/vol, in distilled H2O) and covered with a coverslip.

RESULTS AND DISCUSSION PHB synthase PhaC and polyP kinase Ppk2c are active in vivo and in vitro. The PHB synthase phaC gene and the polyP kinase ppk2c gene were separately cloned in pBBR1MCS2 or pCM62 and expressed in a PHB-free and polyP free mutant background of R. eutropha H16 (∆phaC, ∆ppk-all, see Table 1). As a result, the phaC and ppk2c genes were functionally expressed in the ∆phaC, ∆ppk-all R. eutropha mutant as evident from the restoration of the ability to synthesize PHB granules and polyP granules (Figure 1AD). To determine whether the two polymerase proteins are also active in vitro, the phaC and ppk2c genes were cloned in pET28a and overexpressed in E. coli. Both genes included six histidine codons at the 5´-ends for simplified purification of the gene products. The His6-PhaC and His6-Ppk2c proteins were purified by metal chelate affinity chromatography (Figure S1A, B). Both proteins were active as shown by the ACS Paragon Plus Environment

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formation of PHB or polyP when the proteins were incubated with the respective substrates (3-hydroxybutyryl-coenzyme A or ATP) (Figure 2). The activities of the PHB synthase PhaC and the polyP kinase Ppk2c in the Ppk2c-PhaC fusion were independently confirmed by a colorimetric PHB synthase assay using Ellmann´s reagent or by a toluidine-blue staining of formed polyP, respectively (Figures S2, S3).


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Figure 1: Formation of PHB and polyP by PhaC and Ppk2c in vivo (A-D). Fluorescence microscopy of R. eutropha WT (A). R. eutropha Δppk-all ΔphaC cells with no plasmid (B), with pBBR1MCS2-PphaC-phaC-eyfp (C) or with pCM62-PphaC-ppk2c-mCherry (D). The cells were grown on NB medium supplemented with 0.2% Na-gluconate at 30°C. Note the formation of PHB in (A and C) and of polyP in (A and D), respectively, but neither PHB nor polyP in the control (B).


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Figure 2: Formation of PHB and polyP by purified Ppk2c and purified PhaC. Purified Ppk2c (upper panel) and purified PhaC (lower panel) were incubated with the respective substrates (ATP or 3HB-CoA) for 60 min at 30°C. Subsequently, the samples were stained with DAPI and Nile red and imaged fluorescence microscopically. Granule formation could be observed for both enzymes. Note the absence of any cross-talk between the DAPI-polyP channel and the Nile red-PHB channel. Bars indicate 5µm in all images.

Expression of a ppk2c-phaC fusion restores the formation of polyP and PHB granules in a polyP- and PHB-free background. To investigate whether PHB and polyP could be synthesized by only one polypeptide, a fusion of ppk2c and phaC was constructed and cloned into pBBR1MCS2 under control of the arabinose-inducible PBAD promoter. The recombinant plasmid was transferred by conjugation from E. coli S17-1 to ∆phaC+∆ppkall R. eutropha. When the recombinant strain was cultivated under PHB permissive conditions (NB-medium supplemented with 0.2% sodium gluconate with or without 0.2% arabinose), the formation of polyP and PHB granules was restored. It turned out


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that the addition of arabinose was not essential and polyP and PHB formation was also detected in the absence of arabinose. Apparently, a basal level of ppk2c-phaC expression was independent from the presence of arabinose. As shown in Figure 3, the cells became substantially longer than the control strain (R.


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Figure 3: Fluorescence microscopy of R. eutropha Δppk-all, ΔphaC cells at different time points. Cells harboring pBBR1MCS2-PBAD-ppk2c-phaC were grown on NB medium supplemented with 0.2% Na-gluconate and 0.2% arabinose at 30°C. Samples were stained with DAPI and Nile red. Note abnormal increase of cell length during cultivation and continuous formation of polyP and PHB granules. Effect of polymer granule formation on cell size has been previously noted for R. eutropha37,38. Intriguingly, PHB and polyP granules seemed to be aligned alternately along the longer cell axis. Bars correspond to 5 µm.

eutropha ∆phaC, ∆ppk-all, Figure 1B) and harbored several polyP and PHB granules as evident from staining the cells with DAPI and Nile red. Interestingly, the polyP and PHB granules were located close to each other and were aligned alternately along the longer cell axis. These results confirmed that the Ppk2c and PhaC domains of the Ppk2c-PhaC fusion were both active and catalyzed the formation of polyP and PHB. Quantification of the amount of formed polyP and PHB formed by cells grown on NB-gluconate revealed contents of 17% polyP and 46% PHB, respectively. When ppk2c and phaC were expressed not as a fusion gene (ppk2c-phaC), but as two single genes each cloned on a separate plasmid and both plasmids were transformed to R. eutropha ∆phaC, ∆ppk-all, the recombinant cells also formed PHB granules and polyP granules (Figure 4). However, in this case the two types of polymer granules were not aligned alternately; instead, separate polyP granules and separate clusters of PHB granules were observed in the cells. These results suggest that the polyP and PHB granules formed by the Ppk2c-PhaC fusion were physically attached to each other while the individual Ppk2c and PhaC proteins formed physically separated granules. Since formed PHB granules are covalently attached to the active site of the PhaC domain (Cys31939) and since polyP is bound to polyP kinases12,40, the polyP and PHB granules formed by the Ppk2c-PhaC fusion most likely represent


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a block copolymer (polyP-enzyme-PHB complex). Those polymer molecules that have been (already) released from the enzyme by a termination reaction presumably form blends.


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Figure 4: Plasmid derived formation of polyP and PHB granules in R. eutropha Δppk-all, ΔphaC cells. In a polyP-free and PHB-free background (R. eutropha Δppk-all, ΔphaC) the formation of polyP and PHB granules from the pCM62-ppk2c-mCherry and the pBBR1MCS2-phaC-eyfp plasmid, respectively, was observed. With the help of the two plasmids, the cells were able to produce both types of granules again. However, compared to the ppk2c-phaC fusion, the single genes cloned on separate plasmids created granules in the cells some of which were not physically attached to each other. Bars indicate 5 µm.

Purified His6-Ppk2c-PhaC protein catalyzes the formation of polyP-PHB in vitro. To find additional evidence for the formation of a polyP-PHB block copolymer, we documented the formation of polyP and PHB in vitro. To this end, the purified His6Ppk2c-PhaC protein (Figure S1C) was incubated in the presence of the substrates ATP, 3HB-CoA or a combination of both substrates. When only ATP or only 3HB-CoA were present, only polyP or only PHB were synthesized, respectively, as revealed by DAPIstainable polyP or Nile red-stainable PHB (Figure S4). When both substrates were ACS Paragon Plus Environment

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present, polyP and PHB were formed simultaneously and mixed aggregates of polyP and PHB became visible by fluorescence microscopy (Figure 5). The aggregates were about ≥10 µm in size and consisted of more or less globular structures in the range of 0.5 to 1 µm. The formation of these reaction products was strictly dependent on the presence of the enzyme (Ppk2c-PhaC) and on the substrates (ATP and 3HB-CoA). Interestingly, the major part of the formed reaction products was stained both by DAPI and by Nile red; reaction products that were stained only by DAPI or only by Nile red were also present but were only rarely detected. Since a cross talk between the DAPIpolyP channel and the Nile red-PHB channel was excluded (see Figure 2 and Figure S4), our data indicate that the aggregates consist of both types of polymers (polyP and PHB). These data are in agreement with the formation of a blend of polyP and PHB. PHB and polyP molecules, that had not been released from the Ppk2c-PhaC fusion protein e. g. by a chain transfer reaction, represent polyP-protein-PHB block copolymer molecules. These molecules can interact both with released polyP molecules and PHB molecules and can explain the observed physical aggregations (blends) of polyP and PHB granules. Experiments to separate PHB granules from polyP granules by glycerol density gradient centrifugation failed. In contrast, a white-beige band appeared (layer 2 in Figure S5). Microscopical analysis of this band revealed aggregates that could be stained with Nile red and DAPI suggesting that both polymers (polyP and PHB) were present and were physically attached to each other. Substantial amounts of individual ACS Paragon Plus Environment

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granule-like structures that were stained only with DAPI or only with Nile red were only rarely found in this or in other layers of the gradient.


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Figure 5: Formation of polyP-PHB in vitro. The purified fusion protein Ppk2c-PhaC was incubated with ATP and 3HB-CoA at 30°C. After incubation time of 60 min the samples were stained with DAPI and Nile red and imaged by fluorescence microscopy (each row of images corresponds to a different polymer aggregate). Both polymerase domains of the fusion protein were enzymatically active as revealed by the simultaneous formation of aggregates of polyP and PHB granules. Bars represent 5 µm.

CONCLUSIONS In this work we describe the simultaneous formation of two polymers, polyP and PHB, by a fusion of the polyP kinase Ppk2c with the PHB synthase PhaC of R. eutropha. The PHB synthase of R. eutropha is a very robust enzyme and tolerates the presence of


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additional (artificial) domains fused at different positions within the primary amino acid sequence of PhaC41-44. In our study, the Ppk2c moiety was simply fused to the Nterminus of PhaC. However, fusion of Ppk2c to the C-terminus of PhaC is possible as well and also led to the formation of PHB-polyP (data not shown). We never observed the formation of single Nile red foci (corresponding to PHB only) or of single DAPI-foci (corresponding to polyP only) by the purified Ppk2c-PhaC fusion, when both substrates (ATP, 3HB-CoA) were provided simultaneously. Similarly, we detected the formation of polyP or PHB alone if either ATP or 3HB-CoA were provided as the only substrates, respectively. We assume that in the presence of both substrates a block copolymer of polyP and PHB was formed in which the polyP and PHB blocks are linked via the polymer-attached fusion protein. To the best of our knowledge this is the first description of an enzymatic synthesis of an inorganic-organic block copolymer via a synthetic biology approach. However, depending on the rate of termination of polymer synthesis and the number of reinitiation events of polymerization, a substantial portion of the synthesized PHB and polyP molecules represent blends. Due to the highly different properties of polyP (ionic) and PHB (hydrophobic), the molecules presumably will aggregate in separate granules. This means that the PHB molecule part of the polyP-PHB copolymer molecules aggregates preferentially together with the PHB moieties of other polymer molecules. Vice versa, the polyP parts of the polyP-PHB copolymer molecules aggregate mainly with the polyP moieties. Since each of the individually formed granules likely contains several of the respective blocks of the


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copolymer molecules, individual PHB and polyP granules are physically linked together and can be stained by both dyes (DAPI and Nile red). This is probably the reason why we could hardly detect free PHB granules (without adjacent polyP) or free polyP granules (without adjacent PHB) but mainly detected aggregates of polyP and PHB granules in microscopical images. These aggregates could not be separated physically by density gradient centrifugation and formed a distinct band (Figure S5). We predict that other block copolymers/blends of inorganic polyP and organic polymers (polysaccharides, proteins) can be made in an analog approach. Moreover, the formation of a block copolymer/blend of polyP and nucleic acids (DNA, RNA) should also be possible by the construction of a polyP kinase fused to a nucleic acid polymerase or a polynucleotide kinase.

SUPPORTING INFORMATION Figure S1 (SDS-PAGE of purified proteins), Figure S2 (in vitro PHB synthase assays), Figure S3 (products of in vitro polyP kinase assays in toluidine blue-stained PAGE), Figure S4 (fluorescence microscopy of enzymatically formed PHB and polyP), Figure S5 (separation of in vitro produced polymer by glycerol gradient centrifugation and fluorescence microscopy)

AUTHOR INFORMATION Corresponding Author [email protected]


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Author Contributions The study was designed by D.J. The experiments were performed by J.H. and S.R.. The manuscript was prepared by D.J. and J.H.. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by a grant of the Deutsche Forschungsgemeinschaft to D.J. Notes The authors declare no competing financial interests ACKNOWLEDGMENT We thank Anna Kerber for assistance in some experiments ABBREVIATIONS ATP (GTP), adenosine (guanosine) triphosphate; PHA, polyhydroxyalkanoate; PHB, poly(3-hydroxybutyrate); PPK, polyphosphate kinase; polyP, polyphosphate; SDSPAGE, sodium dodecylsulfate poly(acrylamide) gel electrophoresis; CoA, coenzyme A); 3HB, 3-hydroxybutyrate

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For Table of Contents Use Only

Formation of an organic-inorganic biopolymer: polyhydroxybutyrate-polyphosphate

Jennie C. Hildenbrand, Simone Reinhardt, Dieter Jendrossek

Table of Contents Graphic

Formation of polyP-PHB by a polyP kinase-PHB synthase (Ppk2c-PhaC) fusion protein expressed from a plasmid in a polyP- and PHB-negative background of Ralstonia eutropha (∆phaC, ∆ppk-all). PHB and polyP were stained with Nile red and DAPI, respectively. An overlay image of bright field, Nile-Red-PHB channel and DAPI-polyPchannel is shown. Bar corresponds to 5 µm. ACS Paragon Plus Environment

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Formation of an organic-inorganic biopolymer: polyhydroxybutyrate

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