The resistance of Escherichia coli to oxytetracycline

1969 ◽  
Vol 15 (9) ◽  
pp. 1077-1083 ◽  
Author(s):  
Jerold A. Last ◽  
Kazuo Izaki ◽  
J. F. Snell
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Irreversible Binding ◽  
Resistant Strains ◽  
Growth Of Bacteria

The effects of oxytetracycline on sensitive and resistant strains of Escherichia coli were studied in relation to: (1) growth of bacteria in shaken cultures, (2) incorporation of amino acids into polypeptides by cell-free extracts, (3) binding of aminoacyl-tRNA to ribosomes. Our results support the hypothesis that resistance to the antibiotic is due to impermeability of bacteria to the drug.Evidence is also presented that there is no irreversible binding of oxytetracycline at ribosomal sites involved in protein synthesis.

scholarly journals Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

2019 ◽  
Vol 20 (3) ◽  
pp. 492 ◽  
Author(s):  
Jiro Adachi ◽  
Kazushige Katsura ◽  
Eiko Seki ◽  
Chie Takemoto ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Natural Amino Acids ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

scholarly journals Association of nascent polypeptide with 30S ribosomal subunits

1973 ◽  
Vol 136 (4) ◽  
pp. 859-863 ◽  
Author(s):  
Michael Cannon ◽  
M. Amin A. Mirza ◽  
Margaret L. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trichloroacetic Acid ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Ribosomal Subunits ◽  
Nascent Polypeptide ◽  
Sucrose Gradient Centrifugation ◽  
Crude Extracts

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).

scholarly journals Directed evolution of rRNA improves translation kinetics and recombinant protein yield

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fan Liu ◽  
Siniša Bratulić ◽  
Alan Costello ◽  
Teemu P. Miettinen ◽  
Ahmed H. Badran
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Bacterial Population ◽  
Protein Yield ◽  
Rrna Sequence ◽  
Evolutionary Trajectory ◽  
Noncanonical Amino Acids ◽  
Rate Limiting

AbstractIn bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Ribosome maturation in RCstr and RCrel strains

1973 ◽  
Vol 136 (2) ◽  
pp. 235-247 ◽  
Author(s):  
J. E. M. Midgley ◽  
R. J. Smith
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
High Rate ◽  
Chain Elongation ◽  
Chain Initiation ◽  
Rna Accumulation ◽  
Ribosomal Protein Synthesis ◽  
Nucleotide Bases

When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RCstr and RCrel strains continued to accumulate RNA at rates very close to those in growing controls. The effects of trimethoprim on protein synthesis were studied by using as an experimental basis the rate of maturation of ribosomal particles from RNA-rich precursors. 1. In RCstr cultures given nucleotide bases but no amino acids, RNA accumulation was inhibited because of amino acid lack. However, maturation of ribosomes from their precursors was more severely inhibited than was the synthesis of rRNA. The restraints on protein synthesis were more severe at the level of translation than the transcription of operons specific for the formation of ribosomal proteins. The kinetic delay time in the passage of rRNA from RNA-rich intermediates to the final ribosome products was therefore increased some three- to four-fold. 2. In RCrel cultures in the same conditions, trimethoprim inhibition stopped ribosomal particle synthesis, but rRNA-rich precursors accumulated. 3. If glycine+methionine were also added to inhibited RCstr cultures, RNA accumulation resumed at a high rate. However, ribosomal maturation was still considerably disturbed because of a disproportionate response of the cells in the formation of protein and RNA. 4. With RCrel cultures, addition of the amino acids caused a large increase in the rate of ribosome maturation, though the degree of disproportionation between the rates of rRNA and ribosomal protein synthesis was now identical with that found in RCstr strains. 5. When inhibited RCrel cultures were supplemented, there was still a severe inhibition of protein synthesis at the level of chain initiation, but inaccuracies in the process of polypeptide chain elongation were greatly decreased. This suggests that the effects of the RCrel mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.

Mechanism of action of an inhibitor from Proteus vulgaris on cell-free protein synthesis by Escherichia coli B

1969 ◽  
Vol 15 (2) ◽  
pp. 159-164
Author(s):  
J. J. McEvoy ◽  
W. E. Inniss
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Mechanism Of Action ◽  
Ribonucleic Acid ◽  
Proteus Vulgaris ◽  
Flash Evaporation ◽  
Free System ◽  
Messenger Ribonucleic Acid ◽  
Escherichia Coli B

An inhibitory substance(s) has been found in S-30 fractions from Proteus vulgaris which prevented an Escherichia coli B cell-free system from incorporating a mixture of 14C-amino acids, L-phenylalanine-14C, or L-lysine-14C into protein, as directed by natural messenger ribonucleic acid, polyuridylic acid, or polyadenylic acid respectively. Similar results were obtained when the inhibitor was isolated from S-100 fractions by using dialysis, concentration of the dialysate by flash evaporation, hydrolysis, evaporation to dryness, dissolution to the original volume in distilled water, and neutralization. The effect of the inhibitor on the various individual reactions involved in protein synthesis was examined. No effect was found on the activation of amino acids as determined by the formation of L-phenylalanine-14C hydroxamate isolated chromatographically or by adenosine triphosphate – pyrophosphate exchange. Also no inhibition of L-phenylalanine-14C attachment to transfer ribonucleic acid occurred. However, ribosome-dependent reactions were markedly inhibited. The mechanism of action of the inhibitor appeared to be the prevention of binding of phenylalanyl-transfer ribonucleic acid to the ribosomes.

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

1963 ◽  
Vol 41 (1) ◽  
pp. 455-460 ◽  
Author(s):  
S. J. Webb
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Enzyme System ◽  
Factors Affecting ◽  
E Coli ◽  
A Cell

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.

Inhibition of the induced synthesis of protocatechuate oxygenase by o-nitrobenzoic acid

1970 ◽  
Vol 16 (7) ◽  
pp. 609-614
Author(s):  
Karen F. Montgomery ◽  
Norman N. Durham
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trichloroacetic Acid ◽  
Protocatechuic Acid ◽  
Viable Cell ◽  
Insoluble Fraction ◽  
Nitrobenzoic Acid ◽  
Cell Counts ◽  
Oxygenase Activity

The synthesis of protocatechuate oxygenase (EC. 1.13.1.3), an inducible enzyme, by Pseudomonas fluorescens was inhibited by o-nitrobenzoic acid. The inhibitor did not alter the oxygenase activity of induced cells. Viable cell counts indicated that o-nitrobenzoic acid, in the concentrations used in these experiments, did not kill the cells. The inhibitor decreased the incorporation of L-tryptophan-14C and DL-glutamic-14C acid into the hot trichloroacetic acid insoluble fraction of the cell, but had no effect on the uptake of either radioactive amino acids or the inducer, protocatechuic acid. The synthesis of β-galactosidase by Escherichia coli was also inhibited by o-nitrobenzoic acid. The results establish that one site of o-nitrobenzoic acid inhibition is associated with protein synthesis in the cell.

scholarly journals Association of nascent polypeptide and transfer ribonucleic acid with 30S ribosomal subunits

1973 ◽  
Vol 136 (4) ◽  
pp. 865-869
Author(s):  
M. Amin A. Mirza ◽  
Michael Cannon ◽  
Margaret L. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Radioactive Material ◽  
Considerable Proportion ◽  
Experimental Conditions ◽  
Ribosomal Subunits

1. Crude extracts of Escherichia coli programmed in protein synthesis by endogenous mRNA have incorporated amino acids into protein. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has revealed that 30S ribosomal subunits carry associated radioactive material of which a considerable proportion can be removed from ribosomes by treatment of pre-labelled extracts with puromycin. 2. Gradient analyses of incorporations carried out in the additional presence of added32P-labelled tRNA have indicated that tRNA sediments in the regions of the newly synthesized nascent protein and that both labels are associated with all ribosomal components detected on the gradients under the experimental conditions employed. 3. 30S ribosomal subunits carrying both32P and14C labels have been isolated, disrupted with sodium dodecyl sulphate, and analysed by chromatography on Sephadex G-200 columns. Both labels elute closely together and well away from a tRNA marker analysed under identical conditions. 4. It is proposed that 30S ribosomal subunits, isolated from extracts which have synthesized nascent peptides under the direction of endogenous mRNA, carry associated peptidyl-tRNA.

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

1963 ◽  
Vol 41 (2) ◽  
pp. 455-460 ◽  
Author(s):  
S. J. Webb
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Enzyme System ◽  
Factors Affecting ◽  
E Coli ◽  
A Cell

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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