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Journal of Bacteriology, November 2007, p. 8250-8256, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00752-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

Keiichi Uchino,^1,2 Terumi Saito,² Birgit Gebauer,¹ and Dieter Jendrossek^1*

Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany,¹ Laboratory of Molecular Microbiology, Kanagawa University, Kanagawa 259-1293, Japan²

Received 14 May 2007/ Accepted 10 August 2007

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Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from ¹⁴C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD⁺, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD⁺/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

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Poly(3-hydroxybutyrate) (PHB) is the most prominent member of the bacterial polyhydroxyalkanoates (PHA). PHA are osmotic neutral reservoirs of carbon and energy and are synthesized when more carbon sources are available than can be consumed by the bacteria. PHA are reutilized (mobilized) during times of starvation and greatly enhance the survival of bacteria in the absence of a suitable exogenous carbon source.

PHB is synthesized by condensation of two molecules of acetyl coenzyme A (CoA) to acetoacetyl-CoA (with a thiolase encoded by phaA), subsequent reduction to 3-hydroxybutyryl-CoA (with a reductase encoded by phaB), and polymerization to PHB (with a synthase encoded by phaC). The polymer can be hydrolyzed to 3-hydroxybutyrate by PHB depolymerases (PhaZs). Biosynthesis and biodegradation of PHA have been investigated by many research groups for about three decades, and a series of books and reviews have been published (11, 13, 30, 34, 39-42). PHB exists in two different forms. In vivo PHB granules consist of an amorphous polymer and are covered by a dense layer consisting of mainly proteins (phasins, PHB synthase, PHB depolymerases, and other proteins) (14, 22, 31, 38). Such granules are called native PHB (nPHB) granules and can be isolated in the native form by glycerol density gradient centrifugation (12, 23, 45). Isolated PHB granules that have been treated with solvents or with compounds that remove the surface layer rapidly crystallize and are referred to as denatured PHB granules (23, 24). (For more details on the impact of the biophysical state of the polymer granules on their susceptibility to enzymatic hydrolysis see references 5, 8, and 12.)

Despite the progress made in understanding the function of individual proteins involved in PHA metabolism, the molecular tools and mechanisms with which a cell decides whether it should synthesize or degrade (mobilize) PHB are not known. Several reports have indicated that PHB synthesis and PHB degradation can happen simultaneously in Ralstonia eutropha, the model organism for PHB metabolism (3, 43). The finding that there is constitutive expression of PHB synthase and PHB depolymerases in R. eutropha is in agreement with these findings (20). However, synthesis of PHB by condensation of 3-hydroxybutyryl-CoA (3HB-CoA) monomer units to PHB and free CoA by PHB synthase and simultaneous hydrolysis of PHB to 3-hydroxybutyrate (3HB) by intracellular PHB depolymerase make no physiological sense as this would be a futile cycle and waste energy in the form of hydrolyzed thioester bonds. A convincing explanation for this apparent contradiction cannot be given. The literature contains many reports of the existence of putative intracellular PHA depolymerases in R. eutropha and other bacteria (2, 12, 34). Meanwhile, as many as seven PHB depolymerases and two 3HB oligomer hydrolase genes are thought to be involved in PHB metabolism in R. eutropha (1, 9, 16, 17, 32, 33, 47). The experimental data showing that the gene products are physiologically important intracellular PHB depolymerases are, however, poor. Only for PhaZa1 is involvement in PHB mobilization supported by independent contributions (9, 32, 47). Nevertheless, we were not able to show significant in vitro PHB depolymerase activity with nPHB granules as the substrate and added PhaZa1 (unpublished data). Either PHB depolymerase has no depolymerase activity in vitro under the conditions used or nPHB granules are so densely covered with proteins that excess depolymerase protein cannot bind to the polymer core. We therefore decided to examine nPHB granules as a whole system. For this, we first confirmed that PhaZa1 is a PHB granule-bound protein in vivo by performing a fusion analysis with green fluorescent protein. Isolated nPHB granules from R. eutropha were then examined for various metabolic functions in PHB synthesis and PHB mobilization.

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Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. R. eutropha was grown in nutrient broth (NB) or mineral salts medium with sodium gluconate (or fructose) as described previously at 30°C (1, 5). Escherichia coli strains were maintained in Luria-Bertani broth (37°C). For induction of genes under control of rhamnose or the lac promoter, 0.2% (wt/vol) rhamnose or 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) was added. Sodium acetate (0.4%, wt/vol) was added for synthesis of PHB. Plasmids harboring various combinations of PHB biosynthetic genes (Table 1) were constructed by standard methods. Fusion of phaZa1 with egfp was performed like apdA-egfp fusion (10).

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TABLE 1. Bacterial strains and plasmids

Isolation of PHB granules. nPHB granules were isolated from French press-lysed cells (in the presence of 1 mM dithiothreitol [DTT]) by two glycerol gradient centrifugations as described previously (5, 12, 23). Cholate-coated artificial PHB (aPHB) granules were prepared as described elsewhere (1).

Thiolysis of PHB. A reaction mixture (0.5 ml) containing 250 µl nPHB granules (100 to 200 mg/ml depending on the batch of nPHB granules), 50 mM potassium phosphate buffer (pH 7.0), 1 mM CoA, and 1 mM DTT was incubated at 30°C. At selected time points samples (0.1 ml) were taken, acidified with 10 µl of 1 N HCl, and centrifuged (20,800 x g, 5 min). The supernatant was analyzed by high-performance liquid chromatography (HPLC). In some experiments nPHB granules were replaced by cholate-coated aPHB granules (final concentration, 2.5 mg/ml). Experiments with a cofactor regeneration system were performed in the presence of 0.1 M glucose, 1 mM NAD⁺, and 0.5 U glucose dehydrogenase (220 U/mg) or in the presence of 0.1 M glucose-6-phosphate, 1 mM NADP, and glucose-6-phosphate dehydrogenase (2.5 U).

Synthesis of PHB from acetyl-CoA. A reaction mixture (0.1 ml) containing 10 µl nPHB granules, 50 mM potassium phosphate buffer (pH 7.0), 1 mM DTT, 1 mM NADPH, 0.1 M glucose-6-phosphate, 2.5 U glucose-6-phosphate dehydrogenase, 1 or 2 mM acetyl-CoA, and 0.17 mM [1-¹⁴C]acetyl-CoA (2.18 GBq/mmol; GE Healthcare) was incubated at 30°C for the times indicated below. Samples (50 µl) were acidified with 2.5 µl trichloroacetic acid, and 1 ml of water was added. The precipitate formed after centrifugation (20,800 x g, 5 min) was dissolved in trichloromethane (50°C) and washed three times with 1 ml water. The solvent was evaporated, and the solids were dissolved in 0.1 ml trichloromethane. After addition of 10 ml of scintillation fluid, the radioactivity was determined with a scintillation counter. The radioactivity in the supernatant of the stopped reaction mixture was also determined. A heat-treated control (20 min, 100°C) of nPHB granules served as a control.

HPLC conditions and other methods. One to 5 µl of the supernatant of a reaction mixture was loaded onto a reverse-phase C₁₈ HPLC column (5 µm; 4.6 by 150 mm; Eclipse XDB-C18; Agilent). Samples were eluted at a flow rate of 0.8 ml/min. The buffers were 0.05 M potassium phosphate buffer (pH 4.7) (solution A) and pure methanol (solution B). The elution conditions were as follows: a gradient from 5% solution B to 25% solution B within 25 min, a gradient to 50% solution B within 8 min (33 min), and a gradient to pure solution B within 1 min (34 min). After this, the column was run isocratically with 5% solution B for 5 min (39 min). Solutions of CoA, acetyl-CoA, and 3-HB-CoA (Sigma) served as standards. pH stat experiments were performed with isolated nPHB, and released products were determined as described previously (5). All other experiments were performed by using standard procedures.

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PhaZa1 is a PHB granule-bound protein in vivo. The putative PHB depolymerase gene, phaZa1, was fused to the green fluorescent protein gene, egfp, cloned in broad-host-range vector pBBRMCS2, and transferred to R. eutropha. Transconjugant cells that were grown in NB for a few hours or in mineral salts medium with gluconate contained several PHB granules, as revealed by detection of refractive globular structures by phase-contrast microscopy. The same PHB granules showed green fluorescence (Fig. 1), indicating that the fusion protein was expressed and colocalized with PHB granules. When the cells were stained with Nile red, the granules showed the typical fluorescence of enhanced green fluorescent protein (Egfp) and Nile red (not shown). After 30 h of incubation NB cultures had lost all PHB granules, and green fluorescence could not be detected anymore. We concluded that PhaZa1-Egfp was expressed during phases of PHB accumulation and was associated with PHB granules.

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FIG. 1. Fluorescence microscopy analysis of R. eutropha H16 harboring pBBRMCS2::phaZa1-egfp during exponential growth. Bacteria were visualized with a Zeiss Axioplan fluorescence microscope using an F41-54 Cy2 filter. Pictures were taken under phase-contrast and fluorescence conditions with a digital camera (Coolsnap) and were processed with the Metaview/Metamorph software (Visitron Systems).

Catalytic properties of isolated nPHB granules. Isolated nPHB granules were examined for PHB depolymerase activity by pH stat analysis as described previously (5). Wild-type granules isolated from gluconate-grown cells had a specific acid release rate of 4 nmol min^–1 mg nPHB^–1. When the same experiment was performed with nPHB granules isolated from the R. eutropha phaZa1 mutant, a reduced value, 3 nmol min^–1 mg nPHB^–1, was obtained. However, the acid-releasing activity of nPHB granules varied by a factor of 3 for different batches of isolated nPHB granules, and the significance of the reduced activity of nPHB granules isolated from the phaZa1 mutant is difficult to assess. Product analysis by enzymatic determination of 3HB using 3HB dehydrogenase and by HPLC analysis after derivatization with bromophenacylbromide showed that monomeric 3HB was the only detectable product of in vitro hydrolysis of isolated nPHB granules from the wild type and from the phaZa1 mutant (data not shown). No indication of release of dimers or higher oligomers was obtained. To investigate the catalytic capabilities of nPHB-bound enzymes, nPHB granules isolated from R. eutropha were incubated in the presence of different substrates and potential cofactors. Samples were taken at intervals as indicated below and screened for soluble products by HPLC analysis.

Thiolysis of nPHB. Isolated nPHB granules were incubated in the presence of 1 mM CoA. HPLC analysis of the soluble products showed that the area of the CoA peak decreased from 0.91 to 0.48 mM and that two new peaks appeared (Fig. 2 and Table 2, experiment 1). The two new peaks were identified as 3HB-CoA and acetyl-CoA by comparison with standard compounds. In addition, the masses (m/z values) of the ions at both peaks were determined by HPLC-electrospray ionization (ESI)-mass spectrometry (MS) and corresponded to the expected masses of 3HB-CoA (m/z 854) and acetyl-CoA (m/z 810). Detection of 3HB-CoA and acetyl-CoA strictly depended on the presence of CoA. Interestingly, the size of the acetyl-CoA peak increased with prolonged incubation time of the granules up to 0.21 mM, while the size of the 3HB-CoA peak slightly decreased from 0.29 to 0.24 mM (Fig. 3). This result suggests that nPHB granules are able to cleave PHB via thiolysis and that intermediately formed 3HB-CoA can be converted to acetyl-CoA.

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FIG. 2. HPLC analysis of products formed during thiolysis. nPHB granules isolated from R. eutropha wild-type strain H16 (wt) and from the phaZa1 mutant were incubated in the presence of CoA as described in Materials and Methods. The reaction was stopped by acidification at the times indicated, and 5 µl of the supernatant obtained after centrifugation was analyzed by HPLC. Peaks identified as CoA, acetyl-CoA, and 3HB-CoA by comparison with standards and by HPLC-ESI-MS are indicated.

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TABLE 2. Assay of in vitro thiolysisa

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FIG. 3. Time course of consumption of CoA and formation of acetyl-CoA and 3HB-CoA during thiolysis of wild-type (wt) and phaZa1 mutant nPHB granules as revealed by determination of peak areas after HPLC. The peak area for free CoA at time zero corresponds to 1 mM. The experimental conditions are described in the legend to Fig. 2.

Conversion of 3HB-CoA to acetyl-CoA requires the presence of cofactors, such as NAD⁺ or NADP⁺, which presumably are present at only trace levels in isolated nPHB granules. We therefore repeated the thiolysis experiments in the presence of added 1 mM NAD⁺ or 1 mM NADP⁺ (Table 2, experiments 2 and 3). Peaks corresponding to 0.36 mM acetyl-CoA and 0.22 mM 3HB-CoA were detected in the presence of 1 mM NADP⁺. Apparently, NADP⁺ has no significant effect on product formation. Interestingly, acetyl-CoA (0.33 mM), but no detectable 3HB-CoA, was found in the presence of 1 mM NAD⁺. We assumed that intermediately formed 3HB-CoA is rapidly converted to acetyl-CoA in NAD⁺-dependent reactions so that the concentration of 3HB-CoA is below the detection limit.

Next, we tested the influence of the presence of reduced cofactors [NAD(P)H] on thiolysis and subsequent reactions. To keep the concentration of the reduced cofactors constant and high, we added an NAD(P)H regeneration system to nPHB granules as described in Materials and Methods. Without the regeneration system we detected 0.27 mM 3HB-CoA and traces of acetyl-CoA (0.07 mM) when nPHB granules were incubated with CoA (Table 2, experiment 4a). However, in the presence of (i) the NADH regeneration system (experiment 4b), (ii) the NADPH regeneration system (experiment 4c), or (iii) both regeneration systems (experiment 4d) only 3HB-CoA (0.1 to 0.21 mM) was produced; no acetyl-CoA or only traces (0.03 mM) of acetyl-CoA could be detected. Apparently, high NADH/NAD⁺ or NADPH/NADP⁺ ratios prevented oxidation of the 3HB-CoA formed.

The experiments described above clearly show that isolated nPHB granules can cleave PHB to 3HB-CoA via thiolysis. To identify the enzyme responsible for the thiolysis reaction, we performed experiments with nPHB granules isolated from recombinant E. coli harboring different combinations of pha genes (Table 1). Isolated nPHB granules of strains harboring phaCAB of R. eutropha as the only PHB-related genes did not produce levels of 3HB-CoA in the presence of added CoA that were significantly above the detection limit (Table 2, experiment 5). Thiolysis did also not occur when phaP1 (encoding phasin [29]) or phaZa1 (encoding PHB depolymerase [9, 32, 47]) of R. eutropha was present in E. coli (experiments 6 and 7). Interestingly, when both the phaP1 and phaZa1 genes were present in a phaCAB background, nPHB granules of this strain produced 3HB-CoA (0.21 mM) in the presence of CoA (experiment 8). Significant amounts of acetyl-CoA were not detected when nPHB granules of recombinant E. coli were used, indicating that downstream mobilizing enzymes were absent. We concluded that E. coli nPHB granules are able to perform the thiolysis reaction in the presence of PhaP1 and PhaZa1. However, when the experiment was performed with nPHB granules that had been isolated from an R. eutropha phaP1 deletion mutant, 3HB-CoA (0.13 mM) was detected (experiment 9). Presumably, other phasin proteins of R. eutropha (19, 29) can replace the function of PhaP1. Thiolysis with nPHB granules from an R. eutropha phaZa1 strain (experiment 10) resulted in production of slightly reduced but still significant amounts of 3HB-CoA, indicating that other PHB depolymerases are present in R. eutropha H16 (Fig. 2 and 3). To confirm that PhaZa1 and not PHB synthase is responsible for the thiolysis reaction, aPHB granules that contained no proteins were prepared and were incubated in the presence of CoA and PhaZa1 (experiment 11). Up to 0.21 mM 3HB-CoA was detected with these granules, confirming that PhaZa1 is able to catalyze thiolysis. When PhaZa1 was heated to 90°C for 10 min before the reaction was performed, no 3HB-CoA was formed (not shown).

PHB synthesis by nPHB granules from acetyl-CoA. The experiment described above clearly showed that isolated nPHB granules are able to perform thiolysis of PHB and can catalyze oxidative cleavage of 3HB-CoA to acetyl-CoA. We were interested in whether nPHB granules could also catalyze the reverse reaction, synthesis of PHB from acetyl-CoA. Synthesis of PHB from 3HB-CoA catalyzed by PHB synthase has been repeatedly shown by other workers (4, 6, 35) and therefore was not examined by us. When isolated nPHB granules were incubated in the presence of acetyl-CoA and NAD(P)H, formation of free CoA and a decrease in the acetyl-CoA peak were determined by HPLC analysis (Table 3, experiments 1 and 2). This result indicated either that acetyl-CoA was cleaved to acetate and free CoA or that acetyl-CoA was converted to PHB via 3HB-CoA. The same results were obtained when nPHB granules of the R. eutropha H16 phaZa1 mutant were used, indicating that PhaZa1 is not important for the reaction observed (Table 3, experiments 3 and 4). When the experiment was performed in the presence of an NADPH regeneration system, 3HB-CoA (0.17 mM) was detected in addition to free CoA and a reduced amount of acetyl-CoA (Table 3, experiment 5). The identity of 3HB-CoA was confirmed by HPLC-ESI-MS and determination of the corresponding m/z value, m/z 854. We also detected ions with the m/z value characteristic of acetoacetyl-CoA (m/z 852). These findings indicated that PHB could be synthesized from acetyl-CoA by catalysis with enzymes present in isolated nPHB granules. To exclude the possibility that acetyl-CoA is cleaved to free acetate and CoA, with the latter converted to 3HB-CoA via thiolysis, we repeated the experiment with ¹⁴C-labeled acetyl-CoA. After incubation of nPHB in the presence of [¹⁴C]acetyl-CoA and NADPH, the suspension was centrifuged and the radioactivity of the PHB-containing pellet was determined (Table 4). Significant label (33 to 40 µM) was present in the PHB fraction (experiments 1 and 3). Controls in which nPHB granules had been heat treated prior to incubation showed no label in the polymer fraction (Table 4, experiments 2 and 4). Addition of ¹⁴C-labeled acetyl-CoA to nPHB without incubation (time zero) did not result in detection of radioactivity in the PHB fraction, indicating that nonspecific binding of acetyl-CoA to nPHB granules was not significant. When NADPH was omitted, no radioactivity was detected in the polymer fraction, indicating that the reaction is NADPH dependent (Table 4, experiment 5). We concluded that isolated nPHB granules have the ability to catalyze all reactions leading from acetyl-CoA to PHB.

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TABLE 3. Assay of in vitro synthesis of PHBa

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TABLE 4. Synthesis of PHB from 14C-labeled acetyl-CoA

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Biochemical investigation of intracellular mobilization of PHB was hindered in the past by an inability to detect the PHB depolymerase activity of isolated putative intracellular PHB depolymerases with the natural substrate (nPHB granules). A second reason probably was that researchers interested in intracellular mobilization of PHB were influenced by detailed knowledge concerning extracellular PHB depolymerases (PhaZs) (11, 13). Extracellular PhaZs generally have 3HB (or oligomers of 3HB) as end products, and presumably researchers, including us, expected that intracellular PHB depolymerases also release free acids as products. The finding that isolated nPHB granules have thiolytic activity and release 3HB-CoA suggests that researchers could have looked for the "wrong" activity in the past. Only recently, the presence of thiolytic activity was proposed for PHB synthase (44). However, the observed activity was rather low (about 0.01 mM 3HB-CoA). PhaZa1-catalyzed thiolysis of aPHB granules in the presence of free CoA produced ca. 0.2 mM 3HB-CoA (Table 2, experiment 11), and this confirmed that PhaZa1 is at least one of the important intracellular PhaZs and that 3HB-CoA is a primary mobilization product. We cannot yet judge the importance of the other putative intracellular PhaZs of R. eutropha, but we now have a suitable tool to test individual PHB depolymerases for activity. Investigation of the amino acid sequence of PhaZa1 and site-directed mutagenesis experiments showed that Cys183 is the active site in PhaZa1 (15). The presence of a thiol group is in agreement with the presence of thiolytic activity of PhaZa1. The thiol group is sensitive to oxidation, and therefore addition of reduced compounds (DTT) was necessary in our experiments to keep the nPHB granules in a reduced state. Since PhaZa1 is expressed intracellularly under reduced conditions, oxidation of the thiol group in vivo is unlikely. The finding that isolated nPHB granules release free 3HB at a rate of about 4 nmol min^–1 mg^–1 PHB in vitro may be explained by the fact that hydrolysis of an ester bond in an aqueous environment is highly exergonic and may occur as an in vitro reaction of PHB depolymerase and PHB synthase.

Another unexpected result of this study was the finding that isolated nPHB granules are able to catalyze the conversion of acetyl-CoA to PHB and vice versa. Synthesis of PHB from acetyl-CoA was unequivocally shown by the incorporation of ¹⁴C label from [¹⁴C]acetyl-CoA (Tables 3 and 4), and the mobilization reactions were demonstrated by formation of acetyl-CoA from nPHB in the presence of CoA (Table 2). Under appropriate conditions the same granules catalyzed formation of PHB from acetyl-CoA and degradation of PHB to acetyl-CoA. This means that all activities (thiolase, reductase, synthase, depolymerase) are present in the isolated nPHB granule fraction in vitro. We do not know whether the phaCAB and phaZa1 gene products alone are responsible for the formation of PHB and acetyl-CoA. The R. eutropha genome contains several dozen potential thiolase and reductase isologs (26). At least one ketothiolase has been found to be bound to isolated nPHB granules (29). Data for in vivo localization of enzymes on the surface of nPHB granules exist only for the PHB synthase PhaC (7, 25), for the PHB depolymerase PhaZa1 (Fig. 1), and for phasins and the phasin-related protein ApdA in Rhodospirillum rubrum (10). nPHB granules were isolated by layering a soluble crude extract onto the surface of a glycerol gradient. The PHB granules migrated into the glycerol fraction, while the soluble proteins remained in the surface layer of the gradient. However, we cannot exclude the possibility that some enzymes, such as a reductase and a thioloase, artificially bound to nPHB granules during preparation of the granules and that the finding that there were thiolase and reductase activities in the nPHB granule fraction was an experimental artifact. Lysozme, which is often added during cell lysis processes, has been found to be attached to isolated PHB granules (21). Therefore, it is necessary to investigate in vivo localization of thiolase and reductase enzymes. Unfortunately, the R. eutropha genome contains many thiolase and reductase isologs (26).

If nPHB granules harbor all proteins necessary for the formation and mobilization of the polymer, nPHB is more than just storage material. PHB granules could have at least three functions, (i) synthesis of PHA, (ii) storage of PHA, and (iii) mobilization of PHA. Consequently, PHB granules contain many proteins, including at least four phasin isoenzymes, PHB synthase, an acetoacetyl-CoA thiolase(s), an acetoacetyl-CoA reductase(s), a PHB depolymerase(s), and a regulatory protein, PhaR, which are known to be at least present on PHB granules (26-29, 48). We therefore consider PHA granules subcellular organelles rather than simple storage tanks.

A striking consequence of our findings is that the ability of the cells to simultaneously synthesize and degrade PHB (3, 43) does not lead to a futile cycle. If the primary product of PHB mobilization is 3HB-CoA instead of the free acid, there is no loss of energy, and the results of Doi et al. (3) and Taidi et al. (43) showing simultaneous synthesis and degradation of PHB can now be fully understood. Our experiments performed with different precursor molecules and cofactors suggest that the balance between net biosynthesis and net mobilization is controlled by the concentration of key metabolites. In the future, it will be necessary to perform in vitro experiments with nPHB granules and different combinations and different concentrations of putative key metabolites and to determine the formation of products. The in vitro values obtained should be compared with the concentrations of key metabolites determined with living cells cultured under PHB synthesis and PHB mobilization conditions.

 
We thank M. Günther for construction of the PhaZa1-Egfp fusion, W. Armbruster for performing HPLC-ESI-MS, and J. Altenbuchner for providing pHWG640. We also thank A. Steinbüchel for providing the phaP1 mutant.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to D.J.

 
* Corresponding author. Mailing address: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany. Phone: 49-711-685-65483. Fax: 49-711-685-65725. E-mail: dieter.jendrossek{at}imb.uni-stuttgart.de

Published ahead of print on 24 August 2007.

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1
1. Abe, T., T. Kobayashi, and T. Saito. 2005. Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16. J. Bacteriol. 187:6982-6990.[Abstract/Free Full Text]
2
2. de Eugenio, L. I., P. Garcia, J. M. Luengo, J. M. Sanz, J. S. Roman, J. L. Garcia, and M. A. Prieto. 2007. Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme. J. Biol. Chem. 282:4951-4962.[Abstract/Free Full Text]
3
3. Doi, Y., A. Segawa, Y. Kawaguchi, and M. Kunioka. 1990. Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett. 55:165-169.[Medline]
4
4. Fukui, T., A. Yoshimoto, M. Matsumoto, S. Hosokawa, and T. Saito. 1976. Enzymatic synthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera. Arch. Microbiol. 110:149-156.[CrossRef][Medline]
5
5. Gebauer, B., and D. Jendrossek. 2006. Assay of poly(3-hydroxybutyrate) depolymerase activity and product determination. Appl. Environ. Microbiol. 72:6094-6100.[Abstract/Free Full Text]
6
6. Gerngross, T. U., and D. P. Martin. 1995. Enzyme-catalyzed synthesis of poly[(R)-(–)-3-hydroxybutyrate]: formation of macroscopic granules in vitro. Proc. Natl. Acad. Sci. USA 92:6279-6283.[Abstract/Free Full Text]
7
7. Gerngross, T. U., P. Reilly, J. Stubbe, A. J. Sinskey, and O. P. Peoples. 1993. Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules. J. Bacteriol. 175:5289-5293.[Abstract/Free Full Text]
8
8. Handrick, R., S. Reinhardt, M. L. Focarete, M. Scandola, G. Adamus, M. Kowalczuk, and D. Jendrossek. 2001. A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids. J. Biol. Chem. 276:36215-36224.[Abstract/Free Full Text]
9
9. Handrick, R., S. Reinhardt, and D. Jendrossek. 2000. Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha. J. Bacteriol. 182:5916-5918.[Abstract/Free Full Text]
10
10. Handrick, R., S. Reinhardt, D. Schultheiss, T. Reichart, D. Schüler, V. Jendrossek, and D. Jendrossek. 2004. Unraveling the function of the Rhodospirillum rubrum activator of polyhydroxybutyrate (PHB) degradation: the activator is a PHB-granule-bound protein (phasin). J. Bacteriol. 186:2466-2475.[Abstract/Free Full Text]
11
11. Jendrossek, D. 2002. Extracellular polyhydroxyalkanoate depolymerases: the key enzymes of PHA degradation, p. 41-83. In Y. Doi and A. Steinbüchel (ed.), Biopolymers, vol. 3b. Polyesters II. Wiley-VCH, Weinheim, Germany.[Medline]
12
12. Jendrossek, D. 2007. Peculiarities of PHA granule preparation and PHA depolymerase activity determination. Appl. Microbiol. Biotechnol. 74:1186-1196.[CrossRef][Medline]
13
13. Jendrossek, D., and R. Handrick. 2002. Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56:403-432.[CrossRef][Medline]
14
14. Jendrossek, D., O. Selchow, and M. Hoppert. 2007. PHB granules at the early stages of formation are localized close to the cytoplasmic membrane in Caryophanon latum. Appl. Environ Microbiol. 73:586-593.[Abstract/Free Full Text]
15
15. Kobayashi, T., and T. Saito. 2003. Catalytic triad of intracellular poly(3-hydroxybutyrate) depolymerase (PhaZ1) in Ralstonia eutropha H16. J. Biosci. Bioeng. 96:487-492.[Medline]
16
16. Kobayashi, T., M. Shiraki, T. Abe, A. Sugiyama, and T. Saito. 2003. Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase. J. Bacteriol. 185:3485-3490.[Abstract/Free Full Text]
17
17. Kobayashi, T., K. Uchino, T. Abe, Y. Yamazaki, and T. Saito. 2005. Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16. J. Bacteriol. 187:5129-5135.[Abstract/Free Full Text]
18
18. Kovach, M. E., P. H. Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. Roop II, and K. M. Peterson. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175-176.[CrossRef][Medline]
19
19. Kuchta, K., L. Chi, H. Fuchs, M. Pötter, and A. Steinbüchel. 2007. Studies on the influence of phasins on accumulation and degradation of PHB and nanostructure of PHB granules in Ralstonia eutropha H16. Biomacromolecules 8:657-662.[CrossRef][Medline]
20
20. Lawrence, A. G., J. Schoenheit, A. He, J. Tian, P. Liu, J. Stubbe, and A. J. Sinskey. 2005. Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation. Appl. Microbiol. Biotechnol. 68:663-672.[CrossRef][Medline]
21
21. Liebergesell, M., B. Schmidt, and A. Steinbüchel. 1992. Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D. FEMS Microbiol. Lett. 78:227-232.[CrossRef][Medline]
22
22. Mayer, F., M. H. Madkour, U. Pieper-Fürst, R. Wieczorek, M. Liebergesell, and A. Steinbüchel. 1996. Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies. J. Gen. Microbiol. 42:445-455.
23
23. Merrick, J. M., and M. Doudoroff. 1964. Depolymerization of poly-beta-hydroxybutyrate by intracellular enzyme system. J. Bacteriol. 88:60-71.[Abstract/Free Full Text]
24
24. Merrick, J. M., D. G. Lundgren, and R. M. Pfister. 1965. Morphological changes in poly-beta-hydroxybutyrate granules associated with decreased susceptibility to enzymatic hydrolysis. J. Bacteriol. 89:234-239.[Abstract/Free Full Text]
25
25. Peters, V., and B. H. Rehm. 2005. In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases. FEMS Microbiol. Lett. 248:93-100.[CrossRef][Medline]
26
26. Pohlmann, A., W. F. Fricke, F. Reinecke, B. Kusian, H. Liesegang, R. Cramm, T. Eitinger, C. Ewering, M. Potter, E. Schwartz, A. Strittmatter, I. Voss, G. Gottschalk, A. Steinbuchel, B. Friedrich, and B. Bowien. 2006. Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat. Biotechnol. 24:1257-1262.[CrossRef][Medline]
27
27. Pötter, M., M. H. Madkour, F. Mayer, and A. Steinbüchel. 2002. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148:2413-2426.[Abstract/Free Full Text]
28
28. Pötter, M., H. Müller, and A. Steinbüchel. 2005. Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16. Microbiology 151:825-833.[Abstract/Free Full Text]
29
29. Pötter, M., H. Müller, F. Reinecke, R. Wieczorek, F. Fricke, B. Bowien, B. Friedrich, and A. Steinbüchel. 2004. The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology 150:2301-2311.[Abstract/Free Full Text]
30
30. Pötter, M., and A. Steinbüchel. 2006. Biogenesis and structure of polyhydroxyalkanoate granules, p. 109-136. In J. M. Shively (ed.), Inclusions in prokaryotes, vol. 1. Springer Verlag, Berlin, Germany.
31
31. Pötter, M., and A. Steinbüchel. 2005. Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6:552-560.[CrossRef][Medline]
32
32. Saegusa, H., M. Shiraki, C. Kanai, and T. Saito. 2001. Cloning of an intracellular poly[D(–)-3-hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product. J. Bacteriol. 183:94-100.[Abstract/Free Full Text]
33
33. Saegusa, H., M. Shiraki, and T. Saito. 2002. Cloning of an intracellular D(–)-3-hydroxybutyrate-oligomer hydrolase gene from Ralstonia eutropha H16 and identification of the active site serine residue by site-directed mutagenesis. J. Biosci. Bioeng. 94:106-112.[Medline]
34
34. Saito, T., and T. Kobayashi. 2002. Intracellular degradation of PHAs, p. 23-40. In Y. Doi and A. Steinbüchel (ed.), Biopolymers, vol. 3b. Polyesters II. Wiley-VCH, Weinheim, Germany.
35
35. Schubert, P., A. Steinbuchel, and H. G. Schlegel. 1988. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J. Bacteriol. 170:5837-5847.[Abstract/Free Full Text]
36
36. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784-791.[CrossRef]
37
37. Spiekermann, P., B. H. Rehm, R. Kalscheuer, D. Baumeister, and A. Steinbüchel. 1999. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch. Microbiol. 171:73-80.[CrossRef][Medline]
38
38. Steinbüchel, A., K. Aerts, W. Babel, C. Follner, M. Liebergesell, M. H. Madkour, F. Mayer, U. Pieper-Fürst, A. Pries, H. E. Valentin, et al. 1995. Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can. J. Microbiol. 41(Suppl. 1):94-105.[Medline]
39
39. Steinbüchel, A., and S. Hein. 2001. Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms. Adv. Biochem. Eng. Biotechnol. 71:81.[Medline]
40
40. Steinbüchel, A., and M. Hofrichter. 2001. Biopolymers. Wiley-VCH, Weinheim, Germany.
41
41. Stubbe, J., and J. Tian. 2003. Polyhydroxyalkanoate (PHA) homeostasis: the role of PHA synthase. Nat. Prod. Rep. 20:445-457.[CrossRef][Medline]
42
42. Stubbe, J., J. Tian, A. He, A. J. Sinskey, A. G. Lawrence, and P. Liu. 2005. Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm. Annu. Rev. Biochem. 74:433-480.[CrossRef][Medline]
43
43. Taidi, B., D. Mansfield, and A. J. Anderson. 1995. Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture. FEMS Microbiol. Lett. 129:201-206.
44
44. Uchino, K., and T. Saito. 2006. Thiolysis of poly(3-hydroxybutyrate) with polyhydroxyalkanoate synthase from Ralstonia eutropha. J. Biochem. (Tokyo) 139:615-621.[Abstract/Free Full Text]
45
45. Wieczorek, R., A. Steinbüchel, and B. Schmidt. 1996. Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria. FEMS Microbiol. Lett. 135:23-30.[CrossRef][Medline]
46
46. York, G. M., B. H. Junker, J. A. Stubbe, and A. J. Sinskey. 2001. Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J. Bacteriol. 183:4217-4226.[Abstract/Free Full Text]
47
47. York, G. M., J. Lupberger, J. Tian, A. G. Lawrence, J. Stubbe, and A. J. Sinskey. 2003. Ralstonia eutropha H16 encodes two and possibly three intracellular poly[D-(–)-3-hydroxybutyrate] depolymerase genes. J. Bacteriol. 185:3788-3794.[Abstract/Free Full Text]
48
48. York, G. M., J. Stubbe, and A. J. Sinskey. 2002. The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J. Bacteriol. 184:59-66.[Abstract/Free Full Text]

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Journal of Bacteriology, November 2007, p. 8250-8256, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00752-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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