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 Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway

2002 ◽  
Vol 184 (23) ◽  
pp. 6690-6699 ◽  
Author(s):  
Daisuke Umeno ◽  
Alexander V. Tobias ◽  
Frances H. Arnold
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Substrate Specificity ◽  
Significant Variation ◽  
The Other ◽  
Biosynthetic Pathways ◽  
Product Range

ABSTRACT The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C30 activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway. These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

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 Induction of the Galactose Enzymes in Escherichia coli Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer d-Galactose

2008 ◽  
Vol 190 (24) ◽  
pp. 7932-7938 ◽  
Author(s):  
Sang Jun Lee ◽  
Dale E. A. Lewis ◽  
Sankar Adhya
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Carbon Source ◽  
The Other ◽  
Biosynthetic Pathways ◽  
In Vitro Experiments ◽  
Metabolizing Enzymes ◽  
Optical Configuration

ABSTRACT The two optical forms of aldohexose galactose differing at the C-1 position, α-d-galactose and β-d-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of d-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other d-galactose-metabolizing enzymes are coded by genes that constitute an operon in Escherichia coli. The operon is repressed by the repressor GalR and induced by d-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of d-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of d-galactose. Here we report in vivo and in vitro experiments showing that both α-d-galactose and β-d-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the α configuration inactivate the induction capacity of the sugar, the effect of substitutions in the β configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.

scholarly journals Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon

1995 ◽  
Vol 108 (10) ◽  
pp. 3253-3265 ◽  
Author(s):  
M.F. Pittenger ◽  
A. Kistler ◽  
D.M. Helfman
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Insect Cell ◽  
The Other ◽  
Actin Binding ◽  
Gene Products ◽  
Free Concentration ◽  
Alternatively Spliced ◽  
Beta Gene

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.

scholarly journals Characterization of a rationally engineered phaCAB operon with a hybrid promoter design

2014 ◽  
Author(s):  
Iain Bower ◽  
Bobby Wenqiang Chi ◽  
Matthew Ho Wai Chin ◽  
Sisi Fan ◽  
Margarita Kopniczky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ralstonia Eutropha ◽  
Efficient Production ◽  
Experimental Characterization ◽  
E Coli ◽  
Production Processes ◽  
Hybrid Promoter ◽  
Ralstonia Eutropha H16 ◽  
Carbon Store

Biopolymers, such as poly-3-hydroxy-butyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore more efficient P(3HB) production processes are required. In this study, we describe the model-guided design and experimental characterization of several engineered P(3HB) producing operons. In particular, we describe the characterization of a novel hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. The hybrid phaCAB operon represents a step towards the more efficient production of P(3HB), which has an array of applications from 3D printing to tissue engineering.

Detection of reticuloendothelial-depressing substance in shock

1965 ◽  
Vol 209 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Benjamin Blattberg ◽  
Matthew N. Levy
Keyword(s):  
Escherichia Coli ◽  
Superior Mesenteric Artery ◽  
Whole Blood ◽  
Mesenteric Artery ◽  
The Other ◽  
In Vitro Method ◽  
Carbon Particles ◽  
Normal Rat

In an effort to find an in vitro method to detect the presence of reticuloendothelial-depressing substance (RDS), two tests were devised which measured phagocytic activity. One used carbon particles to measure phagocytosis and the other P32-labeled Escherichia coli. Neither method demonstrated an in vitro difference in granulopectic activity between dog plasmas from sham-operated and hemorrhagic-shock or superior mesenteric artery-occluded (SMAO) animals. An in vivo method was used in which the reticuloendothelial activity of the rat was measured in terms of the rate of clearance of injected carbon particles. Occlusion of the superior mesenteric artery of the rat led to the production of RDS. The RDS could be transferred to and demonstrated in a normal rat by means of SMAO rat whole blood, plasma, and dialysate of plasma, but not in RBC or plasma which had been dialyzed. Sham-operated animals were used as controls.

scholarly journals Independent Regulation of Two Genes in Escherichia coli by Tetracyclines and Tet Repressor Variants

2004 ◽  
Vol 186 (13) ◽  
pp. 4399-4401 ◽  
Author(s):  
Annette Kamionka ◽  
Miriam Sehnal ◽  
Oliver Scholz ◽  
Wolfgang Hillen
Keyword(s):  
Escherichia Coli ◽  
Cross Talk ◽  
Reporter Genes ◽  
The Other ◽  
Regulation System ◽  
The Novel ◽  
Wild Type ◽  
Tet Repressor

ABSTRACT We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

Suppression by the ColV,I-K94 plasmid of inhibitor sensitivities in ompA mutants of Escherichia coli

1988 ◽  
Vol 34 (2) ◽  
pp. 148-156 ◽  
Author(s):  
Claudia F. L. Reakes ◽  
Caroline M. M. Deeney ◽  
Margaret Goodson ◽  
Robin J. Rowbury
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Fusidic Acid ◽  
Polymyxin B ◽  
Aminoglycoside Antibiotics ◽  
The Other ◽  
Gene Products ◽  
Escherichia Coli K12 ◽  
Increased Sensitivity ◽  
Large Plasmids

A series of ompA mutants derived from Escherichia coli K12 strains showed increased sensitivity (compared with the ompA+ parents) to aminoglycoside antibiotics and to other cationic agents including polymyxin B. One tested mutant also showed increased sensitivity to nafcillin and fusidic acid, but not to the hydrophilic ampicillin. All these inhibitor sensitivities in the ompA mutants were suppressed by ColV, I-K94 and by certain other ColV plasmids, but not by any of the other tested large plasmids. Suppression correlated with the production of the VmpA protein, but transfer and colicin components were not needed for suppression. Further comparison of the ompA and vmpA genes and their products was made and it indicated that there is little if any homology between the genes, that the synthesis of their products is regulated by quite different mechanisms, and that regions of these gene products exposed at the cell surface show different susceptibility to protease attack after denaturation.

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.

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