scholarly journals Ralstonia eutropha H16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

2003 ◽  
Vol 185 (13) ◽  
pp. 3788-3794 ◽  
Author(s):  
Gregory M. York ◽  
Joachim Lupberger ◽  
Jiamin Tian ◽  
Adam G. Lawrence ◽  
JoAnne Stubbe ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Complete Sequence ◽  
Inverse Pcr ◽  
Degenerate Primers ◽  
Phb Depolymerase ◽  
Phb Production ◽  
Ralstonia Metallidurans

ABSTRACT Intracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.

Role of gibberellic acid in cotton fibre development

2002 ◽  
Vol 138 (3) ◽  
pp. 255-260 ◽  
Author(s):  
S. J. GOKANI ◽  
V. S. THAKER
Keyword(s):  
Gibberellic Acid ◽  
Fibre Length ◽  
Dry Weight ◽  
Cotton Fibre ◽  
Growth Phases ◽  
Fibre Development ◽  
The Media

Fibres of three cotton cultivars (Gossypium hirsutum H-4, H-8 and G. arboreum G. Cot-15) were analysed for growth in terms of fibre length and dry weight and endogenous gibberellic acid (GA3) content thrice during 1997–2000, at Rajkot. The development of cotton fibre was divided into four distinct growth phases but overlap between elongation and secondary thickening was considerable which suggests that both these phases are independent of each other. During fibre elongation, GA3 content remained low and increased after a decrease in the rate of fibre elongation in all three genotypes. The long staple cultivar (H-4) showed highest endogenous GA3 content followed by the middle one (H-8) and the short staple cultivar (G. Cot-15). In in vitro studies when GA3, NAA or GA3+NAA was supplemented to the media, increase in fibre length of the short staple cultivar was maximum, followed by the middle one and the long staple cultivar. Both in vivo and in vitro findings suggest that GA3 is one of the important factors that determine fibre length.

scholarly journals Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

2014 ◽  
Vol 80 (24) ◽  
pp. 7702-7709 ◽  
Author(s):  
Jessica Eggers ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Ralstonia Eutropha ◽  
Model Organism ◽  
Dry Weight ◽  
Growth Behavior ◽  
Carbon Limitation ◽  
Content Type ◽  
Phb Depolymerase ◽  
E Coli ◽  
The Impact

ABSTRACTThe model organism for polyhydroxybutyrate (PHB) biosynthesis,Ralstonia eutrophaH16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinantEscherichia coliBL21(DE3) strains were used to study the impact of selected PHB depolymerases ofR. eutrophaH16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinantE. coliBL21(DE3) strains were constructed, which harbored a plasmid carrying thephaCABoperon fromR. eutrophaH16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase fromR. eutrophaH16. It is shown in this study that the growth behavior of the respective recombinantE. colistrains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboringphaZ7reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed ifphaZ1was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

scholarly journals Technical-Economical Approach for PHB Production by Ralstonia Eutropha Strain Using Concentrated Vinasse as Carbon Source and Other Biotechnological Applications

2021 ◽  
Author(s):  
Manuella Silverio ◽  
Rosane Piccoli ◽  
João Reis ◽  
José Gregorio Gomez ◽  
Antonio Baptista
Keyword(s):  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Culture Media ◽  
Dry Weight ◽  
Global Market ◽  
Cellular Growth ◽  
Production Costs ◽  
E Coli ◽  
Phb Production ◽  
Threonine Production

Abstract The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v-1 of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v-1) and concentrated (50% and 75% v v-1) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v-1) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L-1 and PHB = 18.6 g L-1, with YX/S = 0.16 g gGLYCEROL-1, YP/S = 0.32 g gGLYCEROL-1 and 0.25 gPHB Lh-1. Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L-1, PHB = 8.3 g L-1 and YX/S = 0.18 g gSUCROSE-1, YP/S = 0.16 g gSUCROSE-1 and 0.12 gPHB Lh-1. Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kgPHB and 2.6 – 7.3 years of payback time.

scholarly journals New Insight into the Role of the PhaP Phasin of Ralstonia eutropha in Promoting Synthesis of Polyhydroxybutyrate

2001 ◽  
Vol 183 (7) ◽  
pp. 2394-2397 ◽  
Author(s):  
Gregory M. York ◽  
JoAnne Stubbe ◽  
Anthony J. Sinskey
Keyword(s):  
Ralstonia Eutropha ◽  
Volume Ratio ◽  
Growth Conditions ◽  
Deletion Strain ◽  
Granule Formation ◽  
Phb Production ◽  
Polyhydroxyalkanoate Synthase ◽  
Low Levels ◽  
Insight Into

ABSTRACT Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.

scholarly journals Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein–deficient Mice

2002 ◽  
Vol 196 (8) ◽  
pp. 1047-1055 ◽  
Author(s):  
Mei Y. Speer ◽  
Marc D. McKee ◽  
Robert E. Guldberg ◽  
Lucy Liaw ◽  
Hsueh-Ying Yang ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Dry Weight ◽  
Inhibitory Effect ◽  
Matrix Gla Protein ◽  
Mutant Mice ◽  
Arterial Calcification ◽  
Ectopic Calcification ◽  
Lineage Markers

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

scholarly journals Spa Contributes to the Virulence of Type 18 Group A Streptococci

2001 ◽  
Vol 69 (5) ◽  
pp. 2943-2949 ◽  
Author(s):  
Duncan G. J. McLellan ◽  
Edna Y. Chiang ◽  
Harry S. Courtney ◽  
David L. Hasty ◽  
Shirley C. Wei ◽  
...  
Keyword(s):  
Human Blood ◽  
Acute Rheumatic Fever ◽  
Protective Antigen ◽  
Surface Protein ◽  
Complete Sequence ◽  
Group A Streptococci ◽  
Protective Antibodies ◽  
Group A

ABSTRACT Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261–1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.

scholarly journals Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

2001 ◽  
Vol 183 (14) ◽  
pp. 4217-4226 ◽  
Author(s):  
Gregory M. York ◽  
Björn H. Junker ◽  
JoAnne Stubbe ◽  
Anthony J. Sinskey
Keyword(s):  
Regulatory Mechanism ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Gene Replacement ◽  
Pha Synthase ◽  
Wild Type ◽  
Phb Production ◽  
Associated Proteins ◽  
Mixed Populations ◽  
In Cells

ABSTRACT 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 aphaP-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 thephaP-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.

scholarly journals Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in Ralstonia eutropha H16

2007 ◽  
Vol 74 (4) ◽  
pp. 1058-1063 ◽  
Author(s):  
Keiichi Uchino ◽  
Terumi Saito ◽  
Dieter Jendrossek
Keyword(s):  
Escherichia Coli ◽  
Direct Evidence ◽  
Ralstonia Eutropha ◽  
Physiological Function ◽  
The Other ◽  
Gene Products ◽  
Phb Depolymerase ◽  
Ralstonia Eutropha H16 ◽  
Null Mutants

ABSTRACT The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes (phaZa1, phaZb, phaZc, and phaZd1) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.

scholarly journals 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

2014 ◽  
Vol 80 (16) ◽  
pp. 4936-4946 ◽  
Author(s):  
Anna Sznajder ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
High Capacity ◽  
Constitutive Expression ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Phb Depolymerase

ABSTRACTThe putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, ofRalstonia eutrophaH16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granulesin vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1or ΔphaP1-phaP5mutant 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 conditionsin 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 ofphaZd1,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 norlacZfusion 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 truein vivofunctions of PhaZd1 and PhaZd2 remain obscure.

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

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