Native PHB Granules Research Articles

Polyhydroxyalkanoates in Bacterial Cells - More Than just Storage Materials

Since polyhydroxyalkanoates (PHAs) belong among the most widespread storage materials within bacteria genus, it can be assumed that the ability of PHAs accumulation represent significant advantage in the natural environments. Of course, probably the most obvious is the possibility to utilize PHAs when external carbon sources are depleted. Nevertheless, it is likely that PHAs play much more complex role in the stress response of bacteria. Therefore, the aim of this work was to investigate possible influence of PHA accumulation in bacteria on physical properties of the cells and their cytoplasm with respect to possible stress survival. Cells of bacteria Cupriavidus necator H16 with various poly(3-hydroxybutyrate) (PHB) content (25 – 91 % of cell dry weight) were subjected to analytical centrifugation employing LUMiSizer. We observed that slope of index instability of bacterial culture increased with rise of PHB content in cells. It indicates that accumulation of PHB granules in bacterial cells affects overall physico-mechanical properties of the cells in particular sedimentation potential and density which might influence their behavior in natural environment such as sedimentation, surface absorption and subsequent biofilm formation. Furthermore, when investigated by Cryo-SEM, the PHB containing cells showed needle-type plastic deformations while these structures were absent in the cells without polymer. This suggests that native intracellular PHB granules reveal completely different mechanical and physico-chemical properties than any other component of bacterial cytoplasm and their flexibility even in deeply-frozen state is significantly higher than that of PHB isolated from bacterial cells. Based on these observations, it can be expected that, aside from their involvement in metabolism, presence of PHB granules dramatically changes physico-mechanical properties of cytoplasm and overall properties of cells which might represent important advantage when cells are exposed to stress conditions.

To be or not to be a poly(3-hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB.

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.

Monitoring the in situ crystallization of native biopolyester granules in Ralstonia eutropha via infrared spectroscopy

Poly(3-hydroxybutyrate) (PHB), a representative polyhydroxyalkanoate (PHA), is a naturally occurring biopolyester stored as tiny, intracellular granules in microbial cells. In vivo, native PHB granules are amorphous, stabilized by a monolayer membrane and intra-granule water. When subjected to varying environmental conditions, the native granules may become partially crystalline. The in situ crystallinity of native PHB granules in Ralstonia eutropha cells suspended in aqueous solution was monitored with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). No sample preparation was required for measurement. A major measurement error could be caused by the evaporation of water. Therefore, the infrared absorption spectra should be taken after the initial settlement of cells, but before excessive dehydration. Background interference caused by water and non-PHB biomass was constant throughout the time course of measurement, regardless of granule crystallinity. The wavenumber 1184 cm −1 was found to be most sensitive to the in situ crystallinity of native PHB granules.

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

Crystallization Kinetics of Poly(3-hydroxybutyrate) Granules in Different Environmental Conditions

Poly(3-hydroxybutyrate) (PHB) is a natural biopolyester accumulated in microbial cells as tiny amorphous granules. The nano- micro-particles have a variety of potential applications and behave differently in different environments. In this work, the in situ crystallization of native PHB granules was investigated under different environmental conditions. The isothermal crystallization kinetics of the granules was shown to follow Avrami’s equation. The model parameter describing crystal growth is a function of temperature or pH and estimated from in situ crystallization measurements with attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Empirical equations describing crystal growth are derived for the parameter values. PHB granules heated at 80-140°C in acidic solution (pH 2) up to 4 hr showed an increase in crystallinity from about 5% to 35% and moderate particle aggregation. PHB granules suspended in alkaline solutions (pH 9-12) at room temperature up to 4 hr showed an increase in crystallinity up to 45% and very little particle aggregation. It was found that the amorphousness of PHB granules in vivo is stabilized by water, lipids and proteins. Upon removal of these impurities, partial crystallization is induced which may inhibit extensive particle aggregation.

Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

The 3-dimensional structure of the Paucimonas lemoignei poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 has significant similarity to Bacillus subtilis lipase LipA but differs from the latter by the presence of an additional domain. Analysis of this lid-like domain revealed the presence of many hydrophobic amino acid residues including Tyr 105. In this study we constructed His-tag fusions of PhaZ7 for simplified purification and investigated the effect of amino acid exchange of eight tyrosine codons of the lid-like domain. Exchanges of Tyr 103, Tyr 172, Tyr 173, Tyr 203 or Tyr 204 to alanine or serine had no phenotype but muteins with substitution of Tyr 189, Tyr 190 and Tyr 105 to alanine showed a lag phase of the in vitro PHB depolymerase reaction. Replacement of Tyr 105 by glutamate further increased the lag phase. Binding assays of the purified PHB depolymerase proteins with the natural substrate, native PHB granules, revealed a significantly reduced binding ability of the Tyr 105Glu mutant compared to the wild type protein and confirmed that Tyr 105 is involved in interaction with the polymeric substrate.

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.

Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable ‘activator’ fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.

Intracellular poly(3-hydroxybutyrate) depolymerase inAlcaligenes eutrophus

The intracellular depolymerization of poly(3-hydroxybutyrate) (PHB) in Alcaligenes eutrophus was investigated. PHB granules of A. eutrophus released D-(−)-3-hydroxybutyrate when incubated at 37 °C. The pH profile of autodigestion of the PHB granules revealed two peaks of activity, one at pH 6–7 and the other at a more alkaline pH. Autodigestion of native PHB granules was inhibited in the presence of Triton X-100. The PHB depolymerase activity was detected in the supernatant from centrifugation at 100 000 × g of the cell extract by using the protease-treated native PHB granules as substrate that lost most of their autodigestive activity. The soluble PHB depolymerase activity increased by about 20-fold or more during PHB synthesis compared with that of PHB-deficient cells, and there was a stringent correlation between PHB content and PHB depolymerase activity in A. eutrophus cells. A soluble PHB depolymerase was partially purified and was inhibited strongly in the presence of diisopropyl fluorophosphate. The pH optimum of the PHB depolymerase in the supernatant fraction was about 8–9. These results indicate that two types of PHB depolymerase may be associated with the native granules, and there was an enzyme with an alkaline pH optimum in the supernatant of the cell extract that seemed to be coregulated with PHB synthesis in the cells.Key words: Alcaligenes eutrophus, poly(3-hydroxybutyrate) depolymerase, poly(3-hydroxybutyrate) granules, biodegradation.

Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules

The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.

Intracellular degradation of poly(3-hydroxybutyrate) granules ofZoogloea ramigeraI-16-M

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d(−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d(−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d(−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of D(−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

1. Summary The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.

MORPHOLOGICAL CHANGES IN POLY-BETA-HYDROXYBUTYRATE GRANULES ASSOCIATED WITH DECREASED SUSCEPTIBILITY TO ENZYMATIC HYDROLYSIS.

Merrick, J. M. (State University of New York, Buffalo), D. G. Lundgren, and R. M. Pfister. Morphological changes in poly-beta-hydroxybutyrate granules associated with decreased susceptibility to enzymatic hydrolysis. J. Bacteriol. 89:234-239. 1965.-A complex enzyme system obtained from extracts of Rhodospirillum rubrum cells hydrolyzes poly-beta-hydroxybutyric acid (PHB) contained in native PHB granules isolated from Bacillus megaterium. A labile factor associated with the granules and necessary for depolymerization is easily destroyed by various chemical and physical treatments. Granules inactivated by these treatments were examined in an electron microscope. In all cases, the distinct morphological appearance of native granules was altered. Morphological changes were mainly characterized by membrane fragmentation, loss of coalescence, and surface alterations. These observations suggest that native PHB granules possess definite structural features, disruption of which results in decreased susceptibility of the polymer to enzymatic hydrolysis.