scholarly journals Improved Production of l-Threonine in Escherichia coli by Use of a DNA Scaffold System

2012 ◽  
Vol 79 (3) ◽  
pp. 774-782 ◽  
Author(s):  
Jun Hyoung Lee ◽  
Suk-Chae Jung ◽  
Le Minh Bui ◽  
Kui Hyeon Kang ◽  
Ji-Joon Song ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Zinc Finger Protein ◽  
Specific Binding ◽  
Double Helix ◽  
Local Concentration ◽  
Simple Diffusion ◽  
Content Type ◽  
Threonine Production ◽  
Dna Double Helix ◽  
Dna Scaffold

ABSTRACTDespite numerous approaches for the development ofl-threonine-producing strains, strain development is still hampered by the intrinsic inefficiency of metabolic reactions caused by simple diffusion and random collisions of enzymes and metabolites. A scaffold system, which can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, was reported to be one of the most promising solutions. Here, we report an improvement inl-threonine production inEscherichia coliusing a DNA scaffold system, in which a zinc finger protein serves as an adapter for the site-specific binding of each enzyme involved inl-threonine production to a precisely ordered location on a DNA double helix to increase the proximity of enzymes and the local concentration of metabolites to maximize production. The optimized DNA scaffold system forl-threonine production significantly increased the efficiency of the threonine biosynthetic pathway inE. coli, substantially reducing the production time forl-threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the host strain by reducing the intracellular concentration of toxic intermediates, such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many useful biomaterials.

scholarly journals (E)-N′-(4-Fluorobenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide

Molbank ◽  
10.3390/m1058 ◽  
2019 ◽  
Vol 2019 (2) ◽  
pp. M1058
Author(s):  
Vinuta Kamat ◽  
Rangappa Santosh ◽  
Suresh Nayak
Keyword(s):  
Escherichia Coli ◽  
Double Helix ◽  
Catalytic Amount ◽  
Target Compound ◽  
Addition Compound ◽  
E Coli ◽  
In Vitro Antibacterial Activity ◽  
Ft Ir ◽  
Dna Double Helix

5-methyl-2-(pyridin-3-yl)-1,3-thiazole-4-carbohydrazide (1) on treatment with 4-fluorobenzaldehyde in presence of catalytic amount of acetic acid, accessed the target compound (2) with the yield of 79%. The target compound was confirmed by 1H-NMR, 13C-NMR, FT-IR and LCMS. In vitro antibacterial activity against Staphylococcus aureus (S. Aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) were carried out and compound 2 showed promising activity against B. subtilis. In addition, compound 2 was analyzed for DNA binding study. It revealed that compound 2 has a promising affinity towards DNA double helix.

scholarly journals Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

2012 ◽  
Vol 78 (7) ◽  
pp. 2179-2189 ◽  
Author(s):  
Makrina Totsika ◽  
Timothy J. Wells ◽  
Christophe Beloin ◽  
Jaione Valle ◽  
Luke P. Allsopp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Specific Binding ◽  
Negative Regulator ◽  
Passenger Domain ◽  
Content Type ◽  
E Coli ◽  
Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

scholarly journals Enhancement of Swimming Speed Leads to a More-Efficient Chemotactic Response to Repellent

2015 ◽  
Vol 82 (4) ◽  
pp. 1205-1214 ◽  
Author(s):  
Richa Karmakar ◽  
R. V. S. Uday Bhaskar ◽  
Rajesh E. Jesudasan ◽  
Mahesh S. Tirumkudulu ◽  
K. V. Venkatesh
Keyword(s):  
Escherichia Coli ◽  
Simple Model ◽  
Drift Velocity ◽  
Concentration Profile ◽  
Swimming Speed ◽  
Intracellular Signaling ◽  
Local Concentration ◽  
Content Type ◽  
Receptor Dynamics ◽  
High Concentrations

ABSTRACTNegative chemotaxis refers to the motion of microorganisms away from regions with high concentrations of chemorepellents. In this study, we set controlled gradients of NiCl2, a chemorepellent, in microchannels to quantify the motion ofEscherichia coliover a broad range of concentrations. The experimental technique measured the motion of the bacteria in space and time and further related the motion to the local concentration profile of the repellent. Results show that the swimming speed of bacteria increases with an increasing concentration of repellent, which in turn enhances the drift velocity. The contribution of the increased swimming speed to the total drift velocity was in the range of 20 to 40%, with the remaining contribution coming from the modulation of the tumble frequency. A simple model that incorporates receptor dynamics, including adaptation, intracellular signaling, and swimming speed variation, was able to qualitatively capture the observed trend in drift velocity.

Technology standardization, competitive behavior, and enterprises’ performance of innovation

2019 ◽  
Vol 38 (1) ◽  
pp. 251-269 ◽  
Author(s):  
Hong Jiang ◽  
Wentao Liu ◽  
Shukuan Zhao ◽  
Yong Chen
Keyword(s):  
Technological Innovation ◽  
Double Helix ◽  
Two Dimensions ◽  
Content Type ◽  
Mediating Role ◽  
Innovation Activities ◽  
Enterprise Innovation ◽  
Double Helix Structure ◽  
Dna Double Helix ◽  
Technology Standardization

Purpose With the development and innovation of IoT technologies, both domestic and international people in the industry believe the development of IoT are the new points of technological innovation and economic growth. But it is becoming more and more prominent that the industries are difficult to connect and the IoT technologies are not standardized. The purpose of this paper is to promote the further development of IoT technology and industry, technology standardization of IoT has become the focus of all concerned parties. Technology standardization, as a technological innovation and development process of the “pruning shears,” has been embedded into the various aspects of innovation activities. Design/methodology/approach Based on many previous theories, the authors establish a matrix of the patterns of technology standardization. The authors use the relative state of the highest level of technology of the enterprise and The degree of independent technological innovation as the two dimensions of the model. At the same time, the authors divide the competitive behaviors into two categories and match them with the technology standardization model. Findings The authors explain the short-term competitive behaviors in the same enterprise and among different enterprises by using the theories of mutualism and competition among species in biology, and make an analogy between the phenomenon of base pairing in DNA double helix structure and the corresponding relationship of two kinds of innovation abilities and two kinds of competitive behaviors in the process of technology standardization. Originality/value Combined with previous theories, explain that innovation capability plays a mediating role in the process in which the combinations of the patterns of technology standardization and competitive behaviors are transformed into the enterprise innovation performance, and the uncertainty of external environment play a regulatory role in the process. Finally, the authors established the final conceptual model for providing theoretical basis for the later research, and put forward the conclusions and prospect at the end of the paper.

scholarly journals Structural insights into the role of architectural proteins in DNA looping deduced from computer simulations

2013 ◽  
Vol 41 (2) ◽  
pp. 559-564 ◽  
Author(s):  
Wilma K. Olson ◽  
Michael A. Grosner ◽  
Luke Czapla ◽  
David Swigon
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Genomic Sequence ◽  
Specific Binding ◽  
Double Helix ◽  
Lac Repressor ◽  
Dna Looping ◽  
Bacterial Gene ◽  
Architectural Proteins ◽  
Dna Double Helix

Bacterial gene expression is regulated by DNA elements that often lie far apart along the genomic sequence, but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, the Escherichia coli Lac repressor protein binds DNA operators, separated by 92 or 401 bp, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as the histone-like (heat-unstable) HU protein, which severely deform the double helix upon binding. In order to gain a better understanding of how the naturally stiff DNA double helix forms the short loops detected in vivo, we have developed new computational methods to study the effects of various non-specific binding proteins on the three-dimensional configurational properties of DNA sequences. The present article surveys the approach that we use to generate ensembles of spatially constrained protein-decorated DNA structures (minicircles and Lac repressor-mediated loops) and presents some of the insights gained from the correspondence between computation and experiment about the potential contributions of architectural and regulatory proteins to DNA looping and gene expression.

scholarly journals Kinetics of dCas9 target search in Escherichia coli

Science ◽  
2017 ◽  
Vol 357 (6358) ◽  
pp. 1420-1424 ◽  
Author(s):  
Daniel Lawson Jones ◽  
Prune Leroy ◽  
Cecilia Unoson ◽  
David Fange ◽  
Vladimir Ćurić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Single Molecule ◽  
Double Helix ◽  
Target Sequence ◽  
Chromosomal Dna ◽  
Guide Rna ◽  
A Cell ◽  
High Concentrations ◽  
Dna Double Helix

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

Protamine-DNA interaction

1978 ◽  
Vol 36 (2) ◽  
pp. 200-201
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Small Volume ◽  
Double Helix ◽  
Dna Interaction ◽  
Dark Field ◽  
Sperm Head ◽  
Nuclear Proteins ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dna Double Helix ◽  
Positively Charged

Dark field electron microscopy has been used for the study of the structure of individual macromolecules with a resolution to at least the 5Å level. The use of this technique has been extended to the investigation of structure of interacting molecules, particularly the interaction between DNA and fish protamine, a class of basic nuclear proteins of molecular weight 4,000 daltons.Protamine, which is synthesized during spermatogenesis, binds to chromatin, displaces the somatic histones and wraps up the DNA to fit into the small volume of the sperm head. It has been proposed that protamine, existing as an extended polypeptide, winds around the minor groove of the DNA double helix, with protamine's positively-charged arginines lining up with the negatively-charged phosphates of DNA. However, viewing protamine as an extended protein is inconsistent with the results obtained in our laboratory.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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