scholarly journals Recombinant production ofZymomonas mobilispyruvate decarboxylase in the haloarchaeonHaloferax volcanii

Archaea ◽  
2005 ◽  
Vol 1 (5) ◽  
pp. 327-334 ◽  
Author(s):  
Steven J. Kaczowka ◽  
Christopher J. Reuter ◽  
Lee A. Talarico ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Stationary Phase ◽  
Zymomonas Mobilis ◽  
Gel Filtration ◽  
Pyruvate Decarboxylase ◽  
Gram Negative Bacteria ◽  
Late Stationary Phase ◽  
E Coli ◽  
Recombinant Production ◽  
Physiological Properties ◽  
Specific Mrna

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene ofZymomonas mobilis(Zmpdc) was expressed inHaloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, includingEscherichia coli. Although the ionic strength of theH. volcaniicytosol differs over 15-fold from that ofE. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like theE. colipurified enzyme, ZmPDC fromH. volcaniicatalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected asH. volcaniitransitioned from log phase to late stationary phase that was inversely proportional to the amount ofpdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.

scholarly journals Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion

1991 ◽  
Vol 276 (2) ◽  
pp. 439-445 ◽  
Author(s):  
R J Diefenbach ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Dipicolinic Acid ◽  
Gel Filtration ◽  
Pyruvate Decarboxylase ◽  
Tryptophan Fluorescence ◽  
Subunit Structure ◽  
Thiamin Diphosphate ◽  
Reversible Binding ◽  
Cofactor Binding ◽  
Series Of Experiments

To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells

Metallomics ◽  
2015 ◽  
Vol 7 (4) ◽  
pp. 683-690 ◽  
Author(s):  
Constanze Pinske ◽  
Frank Sargent ◽  
R. Gary Sawers
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Late Stationary Phase ◽  
E Coli ◽  
Escherichia Coli Cells

The metallochaperone SlyD is essential for nickel delivery to hydrogenase in stationary phaseE. colicells.

The Preparation and Antimicrobial Activity of Peptide Fractions from Blue Mussel (Mytilus edulis) Protein Hydrolysate

2012 ◽  
Vol 485 ◽  
pp. 340-347
Author(s):  
Shi Yuan Dong ◽  
Hong Xia Song ◽  
Yuan Hui Zhao ◽  
Zun Ying Liu ◽  
Bin Bin Wei ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Performance ◽  
Blue Mussel ◽  
Protein Hydrolysate ◽  
Gel Filtration ◽  
Gram Negative Bacteria ◽  
Strong Activity ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Peptide Fractions

The blue mussel protein hydrolysates were separated using the consecutive chromatographic methods including ion exchange, gel filtration, high performance liquid chromatography to identify a potent antimicrobial activity. The fraction (MAMP) separated by HPLC, exhibiting strong activity against Gram-positive (E. coli, P. aeruginosa, S. dysenteriae, P. vuigaris, E. aerogenes) with the minimal inhibitory concentrations (MIC) from 15.63 to 31.25 μg/mL, and Gram-negative bacteria (S. aureus, B. subtilis and M. lysodeikticus) with MIC from 31.25 to 62.5 μg /mL. MAMP had good thermal and pH stability, and consisted of three main amino acids (Ser, Pro and Cys). The antimicrobial activity of MAMP was possibly related to its higher cysteine residues and contents of hydrophobic amino acid. Therefore, MAMP could be a natural antimicrobial source suitable for use as a food additive.

scholarly journals Global Role for ClpP-Containing Proteases in Stationary-Phase Adaptation of Escherichia coli

2003 ◽  
Vol 185 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Dieter Weichart ◽  
Nadine Querfurth ◽  
Mathias Dreger ◽  
Regine Hengge-Aronis
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Growth Phase ◽  
Proteome Analysis ◽  
Resting Cells ◽  
Wild Type ◽  
Late Stationary Phase ◽  
E Coli ◽  
In The Wild ◽  
Dps Protein

ABSTRACT To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.

scholarly journals Muropeptide Rescue in Bacillus subtilis Involves Sequential Hydrolysis by β-N-Acetylglucosaminidase and N-Acetylmuramyl-l-Alanine Amidase

2010 ◽  
Vol 192 (12) ◽  
pp. 3132-3143 ◽  
Author(s):  
Silke Litzinger ◽  
Amanda Duckworth ◽  
Katja Nitzsche ◽  
Christian Risinger ◽  
Valentin Wittmann ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Particulate Material ◽  
Late Stationary Phase ◽  
Phosphotransferase System ◽  
E Coli ◽  
A Cell ◽  
Recycling Pathway ◽  
Hydrolysis Of

ABSTRACT We identified a pathway in Bacillus subtilis that is used for recovery of N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) peptides (muropeptides) derived from the peptidoglycan of the cell wall. This pathway is encoded by a cluster of six genes, the first three of which are orthologs of Escherichia coli genes involved in N-acetylmuramic acid dissimilation and encode a MurNAc-6-phosphate etherase (MurQ), a MurNAc-6-phosphate-specific transcriptional regulator (MurR), and a MurNAc-specific phosphotransferase system (MurP). Here we characterized two other genes of this cluster. The first gene was shown to encode a cell wall-associated β-N-acetylglucosaminidase (NagZ, formerly YbbD) that cleaves the terminal nonreducing N-acetylglucosamine of muropeptides and also accepts chromogenic or fluorogenic β-N-acetylglucosaminides. The second gene was shown to encode an amidase (AmiE, formerly YbbE) that hydrolyzes the N-acetylmuramyl-l-Ala bond of MurNAc peptides but not this bond of muropeptides. Hence, AmiE requires NagZ, and in conjunction these enzymes liberate MurNAc by sequential hydrolysis of muropeptides. NagZ expression was induced at late exponential phase, and it was 6-fold higher in stationary phase. NagZ is noncovalently associated with lysozyme-degradable particulate material and can be released from it with salt. A nagZ mutant accumulates muropeptides in the spent medium and displays a lytic phenotype in late stationary phase. The evidence for a muropeptide catabolic pathway presented here is the first evidence for cell wall recovery in a Gram-positive organism, and this pathway is distinct from the cell wall recycling pathway of E. coli and other Gram-negative bacteria.

Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid

1999 ◽  
Vol 181 (20) ◽  
pp. 6361-6370 ◽  
Author(s):  
Talukder Ali Azam ◽  
Akira Iwata ◽  
Akiko Nishimura ◽  
Susumu Ueda ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Stationary Phase ◽  
Binding Protein ◽  
Protein A ◽  
Dna Binding Protein ◽  
Host Factor ◽  
Late Stationary Phase ◽  
E Coli ◽  
Curved Dna

ABSTRACT The genome DNA of Escherichia coli is associated with about 10 DNA-binding structural proteins, altogether forming the nucleoid. The nucleoid proteins play some functional roles, besides their structural roles, in the global regulation of such essential DNA functions as replication, recombination, and transcription. Using a quantitative Western blot method, we have performed for the first time a systematic determination of the intracellular concentrations of 12 species of the nucleoid protein in E. coli W3110, including CbpA (curved DNA-binding protein A), CbpB (curved DNA-binding protein B, also known as Rob [right origin binding protein]), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Qβ), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor oftd mutant phenotype A). Intracellular protein levels reach a maximum at the growing phase for nine proteins, CbpB (Rob), DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp, and StpA, which may play regulatory roles in DNA replication and/or transcription of the growth-related genes. In descending order, the level of accumulation, calculated in monomers, in growing E. coli cells is Fis, Hfq, HU, StpA, H-NS, IHF*, CbpB (Rob), Dps*, Lrp, DnaA, IciA, and CbpA* (stars represent the stationary-phase proteins). The order of abundance, in descending order, in the early stationary phase is Dps*, IHF*, HU, Hfq, H-NS, StpA, CbpB (Rob), DnaA, Lrp, IciA, CbpA, and Fis, while that in the late stationary phase is Dps*, IHF*, Hfq, HU, CbpA*, StpA, H-NS, CbpB (Rob), DnaA, Lrp, IciA, and Fis. Thus, the major protein components of the nucleoid change from Fis and HU in the growing phase to Dps in the stationary phase. The curved DNA-binding protein, CbpA, appears only in the late stationary phase. These changes in the composition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the genome functions.

scholarly journals Gold(I)-catalysed synthesis of a furan analogue of thiamine pyrophosphate

2014 ◽  
Vol 10 ◽  
pp. 2580-2585 ◽  
Author(s):  
Amjid Iqbal ◽  
El-Habib Sahraoui ◽  
Finian J Leeper
Keyword(s):  
Zymomonas Mobilis ◽  
Furan Ring ◽  
Pyruvate Decarboxylase ◽  
Thiamine Pyrophosphate ◽  
Reaction Intermediates ◽  
Strong Inhibitor ◽  
E Coli ◽  
His Tag ◽  
Efficient Route ◽  
Thiazolium Ring

An analogue of thiamine having a furan ring in place of the thiazolium ring has been synthesised by a short and efficient route, involving gold(I)-catalysed cyclisation of an alkynyl alcohol to form the furan ring. The furan analogue of thiamine diphosphate (ThDP) was also made and tested for binding to and inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis (overexpressed in E. coli with a N-terminal His-tag). It is a very strong inhibitor, with a K i value of 32.5 pM. It was also shown that the furan analogue of thiamine can be functionalised at the C-2 position, which will allow access to mimics of reaction intermediates of various ThDP-dependent enzymes.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

DECONTAMINATION OF VETERINARY SURVEILLANCE OBJECTS BY NEW GENERATION MEDICINE

1970 ◽  
pp. 64-67
Author(s):  
М. S. Saypullaev ◽  
А. U. Koychuev ◽  
Т. B. Mirzoeva
Keyword(s):  
Staphylococcus Aureus ◽  
Inhalation Exposure ◽  
Gram Negative Bacteria ◽  
Hazard Class ◽  
E Coli ◽  
Highly Efficient ◽  
Mucous Membranes ◽  
Environmentally Safe ◽  
New Generation ◽  
And Staphylococcus Aureus

The successful conduct of disinfection measures largely depends on the availability of veterinary practice a highly efficient, environmentally safe disinfectants. In this regard, finding new highly efficient disinfectant remains relevant. Studies found that the "Polied" (OOO "Razvitie XXI Vek, Russia) can be attributed to the highly efficient and environmentally friendly means. Solutions "Polied" have a high disinfectant activity against smooth and rough surfaces in the laboratory against gram-positive, gram-negative bacteria, mycobacteria and spores of microorganisms. Studies have established that solutions should be "Polied" obezzarajivatmi E. coli (EA 1257) concentrations of 0.1% on smooth surfaces and Staphylococcus aureus concentration of 0.05% in 1 hour from the calculation of 0.25-0.3 litres/m2. Disinfection of rough test surfaces against Escherichia coli and Staphylococcus aureus occurred after treatment with 0,3% solution of 3-hour exposure, at a rate of 0.5 l/m2. It was also found that 1.0% solution "Polied" fully obezzarazhivatel test the surface of mycobacteria (PCs-5) and at double the 0.6% concentration for 24 hours. Disinfection of rough test surfaces contaminated with spores of B. cereus (PCs 96) was achieved with a 4.0% solution at twice the irrigation rate of 0.5 l/m2 at an exposure of 24 hours. Toxicity solutions of the drug "Polied" refer to "moderate" threat (hazard class 3) and low-hazard substances (4 hazard class) when applied to the skin, mucous membranes of the eyes, and inhalation exposure on the respiratory system.

Sign in / Sign up

Export Citation Format

Share Document