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The effect of trimethoprim on macromolecular synthesis in Escherichia coli.

ABSTRACT: In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.

SUBMITTER: Smith RJ

PROVIDER: S-EPMC1165948 | biostudies-other | 1973 Oct

REPOSITORIES: biostudies-other

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Project description:When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RC(str) and RC(rel) 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 RC(str) 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 RC(rel) 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 RC(str) 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 RC(rel) 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 RC(str) strains. 5. When inhibited RC(rel) 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 RC(rel) mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.

Project description:During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.

Project description:Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.

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The effect of trimethoprim on macromolecular synthesis in Escherichia coli.

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