FACTORS AFFECTING THE VIABILITY OF AIR-BORNE BACTERIA: VI. THE ACTION OF INOSITOL ON LACTOSE OXIDATION BY DESICCATED ESCHERICHIA COLI

The effect of desiccation on the ability of Escherichia coli to oxidize lactose has been studied. The enzyme system responsible for lactose oxidation is adaptive in E. coli, and amino acids are required for its synthesis. Of the amino acids required, glutamic and aspartic acids proved to be the most active in stimulating the synthesis of the enzymes. The cells of E. coli lose their ability to oxidize lactose on desiccation but if drying is carried out in the presence of i-inositol this loss is prevented. The findings add support to the hypothesis that the death of a cell on desiccation results from the destruction of a component concerned in protein synthesis.

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MICROBIOLOGICAL STUDIES ON THE MECHANISM OF ACTION OF SPARSOMYCIN

Canadian Journal of Microbiology ◽

10.1139/m66-085 ◽

1966 ◽

Vol 12 (4) ◽

pp. 595-604 ◽

Cited By ~ 3

Author(s):

Edward R. Bannister ◽

Dale E. Hunt ◽

Robert F. Pittillo

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Synthesis ◽

Mechanism Of Action ◽

Rna Synthesis ◽

Primary Site ◽

Cross Resistance ◽

E Coli

A primary site of sparsomycin attack in Escherichia coli appears to be inhibition of synthesis of protein, which occurs at concentrations of sparsomycin that do not affect DNA or RNA synthesis. Sparsomycin interferes with the normal excretion of amino acids by E. coli. Some cross-resistance was observed between a culture resistant to sparsomycin and cultures resistant to other inhibitors of protein synthesis.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

BioTech ◽

10.3390/biotech10040024 ◽

2021 ◽

Vol 10 (4) ◽

pp. 24

Author(s):

Marina Snapyan ◽

Sylvain Robin ◽

Garabet Yeretssian ◽

Michèle Lecocq ◽

Frédéric Marc ◽

...

Keyword(s):

Protein Synthesis ◽

Genomic Sequence ◽

Synthetic Activity ◽

Translation System ◽

Free System ◽

Transcription Terminator ◽

Cell Free System ◽

E Coli ◽

Bacterial Transcription ◽

A Cell

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

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Studies on the Incorporation of Amino Acids by a Cell-Free System Obtained from Beef Retina

Canadian Journal of Biochemistry ◽

10.1139/o72-080 ◽

1972 ◽

Vol 50 (5) ◽

pp. 581-587 ◽

Cited By ~ 2

Author(s):

Y. Matuk

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Incubation Medium ◽

Optimum Concentration ◽

Free System ◽

Cell Free System ◽

A Cell

The incorporation of 14C-leucine into proteins by a cell-free system from beef retina was studied. It was found that the optimum concentration of ATP depended on the concentration of ribosomes in the incubation medium. Very little incorporation of 14C-leucine was observed in the absence of K+. The optimum concentration of phosphocreatine required for incorporation of radioactive leucine depended on the concentration of Mg2+ in the incubation medium, and the optimum concentration of K+ appears to be independent of the concentrations of Mg2+ and phosphocreatine used.Retinol and retinal had no effect, but ethanol markedly inhibited protein synthesis at concentrations higher than 2%.Puromycin (10−4 M) inhibited incorporation of 14C-leucine by about 80%. The degree of inhibition by cycloheximide depended on the concentration of pH 5 fraction in the incubation medium.

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Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽

10.1242/dev.24.1.109 ◽

1970 ◽

Vol 24 (1) ◽

pp. 109-118

Author(s):

E. L. Triplett ◽

R. Herzog ◽

L. P. Russell

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Enzymic Activity ◽

Antigenic Determinants ◽

Free System ◽

A Cell ◽

Generating System ◽

Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

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Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

Current Biotechnology ◽

10.2174/2211550108666190418125138 ◽

2019 ◽

Vol 8 (1) ◽

pp. 15-23

Author(s):

Takashi Nakamura ◽

Emi Takeda ◽

Tomoko Kiryu ◽

Kentaro Mori ◽

Miyu Ohori ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Industrial Production ◽

Codon Optimization ◽

Scale Production ◽

Enzymatic Production ◽

Hyperthermophilic Archaeon ◽

E Coli ◽

Aeropyrum Pernix ◽

Natural Amino Acids

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

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Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

Author(s):

Sviatlana Smolskaya ◽

Yaroslav Andreev

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Nonsense Suppression ◽

Expression Vectors ◽

Site Specific ◽

Suppressor Trna ◽

E Coli ◽

Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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