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PubMed1999

Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region.

The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated. Four beta-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding L-aspartate-1-decarboxylase (EC 4.1.1.15), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS- led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encoded L-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of pan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]beta-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha.

Acyltransferases↗
PubMed2001

Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

Acyltransferases↗
PubMed2005

Kinetic studies of polyhydroxybutyrate granule formation in Wautersia eutropha H16 by transmission electron microscopy.

Wautersia eutropha, formerly known as Ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (PHB) as insoluble granules inside the cell when nutrients other than carbon are limited. In this paper, we report findings from kinetic studies of granule formation and degradation in W. eutropha H16 obtained using transmission electron microscopy (TEM). In nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the first time. At the center of the cells, dark-stained "mediation elements" with small granules attached were observed. These mediation elements are proposed to serve as nucleation sites for granule initiation. TEM images also revealed that when W. eutropha cells were introduced into nitrogen-limited medium from nutrient-rich medium, the cell size increased two- to threefold, and the cells underwent additional volume changes during growth. Unbiased stereology was used to analyze the two-dimensional TEM images, from which the average volume of a W. eutropha H16 cell and the total surface area of granules per cell in nutrient-rich and PHB production media were obtained. These parameters were essential in the calculation of the concentration of proteins involved in PHB formation and utilization and their changes with time. The extent of protein coverage of the granule surface area is presented in the accompanying paper.

Culture Media↗
PubMed2005

Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western analysis and transmission electron microscopy.

Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1(a), PhaZ1(b), and PhaZ1(c) (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1(a) were present simultaneously. No PhaZ1(b) or PhaZ1(c) was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1(a). The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (10(6) Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was approximately 60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and deltaphaR strains in TSB provided further support for an alternative mechanism of granule formation.

Bacterial Proteins↗
PubMed1997

Rifampicin carrying polyhydroxybutyrate microspheres as a potential chemoembolization agent.

In this study, we attempted to prepare microspheres from a microbial biodegradable polyester, i.e. polyhydroxybutyrate (PHB) as a potential chemoembolization agent. The drug loaded PHB microspheres were prepared by a solvent evaporation technique, in which methylene chloride, distilled water, and polyvinyl alcohol were utilized as the solvent, dispersion medium, and emulsifier, respectively. Microspheres were obtained within a size range of 5-100 microns by changing the initial polymer/solvent ratio, emulsifier concentration, stirring rate, and initial drug concentration. It was possible to obtain PHB with very narrow size distributions by applying gravity field-flow fractionation technique. Very high drug loadings of up to 407.6 mg rifampicin/g PHB were achieved. Drug release rates were very rapid. Almost 90% of the drug loaded was released in about 24 h. Both the size and drug content of PHB microspheres were found to be effective in controlling the drug release from these microspheres.

Antibiotics, Antitubercular↗
PubMed2000

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate.

Phosphotransacetylase (Pta) catalyzes the reversible conversion of acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.

Acyltransferases↗
PubMed2000

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient.

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia eutropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal sigma 70 promotor. The pKS/PHB was the most efficient plasmid for phb operon expression among the three plasmids used: i.e., pKS-, pAED4, and pJM9131. It was observed that isopropyl-beta-D-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkanoate content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.

Cupriavidus necator↗
PubMed2000

Polyhydroxybutyrate production from carbon dioxide by cyanobacteria.

Genetic characterization and enhancement of polyhydroxybutyrate (PHB) accumulation in cyanobacteria were investigated for efficient PHB production from CO2. The genome DNAs in the PHB-accumulating strains Synechococcus sp. MA19 and Spirulina platensis NIES46 retained the highly homologous region to phaC of Synechocystis PCC6803, whereas low homology was detected in the nonaccumulating strains Synechococcus sp. PCC7942 and Anabaena cylindrica NIES19. Synechococcus sp. MA19, which accumulates PHB up to 30% of dry cell weight from CO2 as the sole carbon source, was mutated by insertion of transposon Tn5 to enhance the PHB accumulation. Genetic and physiological analysis of the mutant indicated that decreased phosphotransacetylase activity could trigger an increase of acetyl coenzyme A leading to enhancement of PHB accumulation. PHB synthase in Synechococcus sp. MA19 was probably attached to thylakoid membrane since PHB granules were associated with pigments. A genetically engineered cyanobacteria retaining soluble PHB synthase from Ralstonia eutropha accumulated pigment-free PHB granules, which is an advantage for the purification of PHB.

Acyltransferases↗
PubMedDate not supplied

[Kinetic parameters of a culture of the hydrogen-oxidizing Ralstonia eutropha, grown under the regimen of biosynthesis of polyhydroxybutyrate].

Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutropha, grown on a gas substrate under the conditions favoring autotrophic biosynthesis of polyhydroxybutyrate, were studied. The following parameters, making it possible to control and optimize the process in industrial situations, were determined: specific rate of substrate consumption, physical properties of culture medium, and coefficients of heat emission and mass transfer.

Culture Media↗
PubMed2004

Surface properties and microporosity of polyhydroxybutyrate under scanning electron microscopy.

This study was designed to investigate the surface properties especially surface porosity of polyhydroxybutyrate (PHB) using scanning electron microscopy. PHB granules were sprinkled on the double-sided sticky tape attached on a SEM aluminium stub and sputtered with gold(10nm thickness) in a Polaron SC515 Coater, following which the samples were placed into the SEM specimen chamber for viewing and recording. Scanning electron micrographs with different magnification of PHB surface revealed multiple pores with different sizes.

Biocompatible Materials↗
PubMed1997

Purification and characterization of citrate synthase from Methylobacterium extorquens--a methylotrophic producer of polyhydroxybutyrate.

Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating; EC 4.1.3.7, CS) was isolated and purified to homogeneity from a methylotrophic producer of polyhydroxybutyrate (PHB), Methylobacterium extorquens 15. The purification procedure includes streptomycin sulfate treatment of cell-free extract, ammonium sulfate fractionation, two steps of hydrophobic chromatography, and ion-exchange chromatography. The specific activity of the final enzyme preparation was 24 U/mg protein. The enzyme has apparent molecular weight 260 kD and consists of four 66-kD subunits. The enzyme shows a sigmoid saturation curve with CoASA (h = 1.3). Kinetic parameters are: K(m) = 84 microM for CoASA; K(m) = 12 microM for oxaloacetate; Vmax = 29.7 mumoles/min per mg protein. KCl at concentrations up to 80 mM activates the CS. ATP exerts a significant inhibitory effect on the enzyme activity, whereas NAD(P)H, isocitrate, alpha-ketoglutarate, ADP, acetoacetyl-CoA, glyoxylate, and glutamate have no influence. A possible role of the CS in coordinated control of CoASA transformation through the tricarboxylic acid cycle and PHB biosynthesis in this methylotroph is discussed.

Chromatography, Liquid↗
PubMed1988

Polyhydroxybutyrate: an intriguing biopolymer.

The microbial polymer poly-3-hydroxybutyrate (PHB) and related poly-hydroxyalkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are unique biodegradable thermoplastics of considerable commercial importance. The structure, properties and regulation of synthesis and degradation of PHB are reviewed and the microbial production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, with properties varying according to copolymer composition, is discussed.

Azotobacter↗
PubMed2002

Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56.

Some members of the archaebacterial family Halobacteriaceae have been determined to accumulate polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate) (PHB). The extremely halophilic archaebacterium strain 56 is capable of accumulating large amounts of PHB. Since measurements of enzyme activities related to archaebacterial PHB biosynthesis have never been achieved, we investigated the enzymology of PHB biosynthesis in strain 56. Crude extracts of strain 56 cultivated under accumulating conditions showed PHB synthase activity, whereas neither beta-ketothiolase nor NADH/NADPH-dependent acetoacetyl-CoA reductase activity was detectable. An 80-kDa protein, cross-reacting with the anti-PHB synthase antibodies raised against the PHB synthase from Ralstonia eutropha, was identified in the crude extract and was strongly enriched by purification of PHB granules. The granule-associated PHB synthase was enzymologically characterized. Enzyme kinetics showed a specific activity of about 4.6U/mg and Hill plot analysis revealed a K(0.5) of 56 microM with (R)-3-hydroxybutyryl-CoA employed as substrate. A Hill coefficient of 1.75 indicated that the PHB synthase exhibited positive cooperativity. The thioesters 3-hydroxyvaleryl-CoA, 4-hydroxybutyryl-CoA, and 3-hydroxydecanoyl-CoA were not accepted as substrates. Moreover, the PHB synthase was found to be competitively inhibited by CoA, showing an IC(50) of 160 microM. The PHB synthase was stable up to 60 degrees C and still exhibited about 90% of the maximum enzyme activity, which was obtained at 40 degrees C. In contrast to the soluble PHB synthase, the granule-bound PHB synthase was almost independent of the salt concentration. The PHB synthase could not be released from the PHB granules, indicating a covalent attachment to the PHB core. This is the first description of an archaebacterial PHA synthase.

Acyltransferases↗
PubMed2001

Analysis of adsorption function of polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1 by using a quartz crystal microbalance.

Enzymatic adsorption and degradation of three types of aliphatic polyester films and two types of polyolefin films by the extracellular PHB depolymerase from Alcaligenes faecalis T1 have been studied by using a quartz crystal microbalance (QCM) technique. Hydrolysis of poly[(R)-3-hydroxybutyrate] was quantitatively followed by the QCM technique. Adsorption of the enzymes to films was also quantitatively detected by the QCM. Kinetic study on enzymatic adsorption suggests that the PHB depolymerase binds to the substrates not only by hydrophobic interaction but also by specific interaction between the ester bonds of polyesters and the binding domain of the enzyme. The results show that the QCM technique is a sensitive tool to study enzymatic degradation kinetics of biodegradable polyesters.

Adsorption↗
PubMed2003

In vivo response to HA-polyhydroxybutyrate/polyhydroxyvalerate composite.

This study examined the morphological and compositional structure of bone-implant interfaces after in vivo implantation into the tibias of rabbits. The implants were composed of biodegradable polyhydroxybutyrate/polyhydroxyvalerate copolymer reinforced with synthetic hydroxyapatite (HA) particles. Optical and scanning electron microscopy techniques were used, including energy-dispersive X-ray analysis. The interface was found to be morphologically, biologically and chemically active throughout the period of study. There was a strong tendency to rebuild the bone structure at the interface after implantation, independent of the composition of the implant, but direct bone bonding with the implant depended on the bioactive nature of the interface, as represented by the HA particles. At all implantation times, lamellar bone formed at the interface and replaced degrading polymer matrix, while engulfing HA filler particles. In regions about 50-100 from the interface, the bone region displayed an osteon organization. Osteoblasts and osteocytes were identified throughout the interface region. The thickness of the newly formed bone significantly increased over the period of the experiment from about 130 microm at 1 month to about 770 microm at 6 months. Materials that behave in this manner may be useful in some bone replacement therapies.

Animals↗
PubMed2004

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha.

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20.0, 20.2, 19.6 and 20.2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

Bacterial Proteins↗
PubMed2006

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding.

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

Amino Acid Motifs↗