scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by ‘magic spot’ nucleotides

1973 ◽  
Vol 136 (2) ◽  
pp. 249-257 ◽  
Author(s):  
R. J. Smith ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Rna Synthesis ◽  
Control Mechanism ◽  
Acid Synthesis ◽  
Macromolecular Synthesis ◽  
Guanosine Tetraphosphate ◽  
Additional Control ◽  
High Concentrations ◽  
Rna Accumulation ◽  
Rate Of Accumulation

During the inhibition of RCstr, but not RCrel 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 RCstr 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 RCstr cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RCstr 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 RCstr 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.

scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

1973 ◽  
Vol 136 (2) ◽  
pp. 225-234 ◽  
Author(s):  
R. J. Smith ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Rna Synthesis ◽  
Protein Chain ◽  
Macromolecular Synthesis ◽  
Chain Initiation ◽  
Chain Polymerization ◽  
Rna Accumulation ◽  
New Protein ◽  
Endogenous Synthesis

In trimethoprim-inhibited RCstr 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. RCrel 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, RCstr 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 RCrel 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 RCstr 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 RCstr or RCrel bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.

scholarly journals The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

1972 ◽  
Vol 128 (5) ◽  
pp. 1007-1020 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterogeneous Material ◽  
Acid Synthesis ◽  
Sucrose Density Gradient ◽  
23S Rrna ◽  
Ribonucleic Acids ◽  
Mrna Content ◽  
High Concentrations ◽  
The Stability

The biosynthesis and stability of various RNA fractions was studied in RCstr and RCrel multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RCstr mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA–RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60–70% of the labelled RNA formed in RCstr mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RCrel strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RCrel strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RCstr or RCrel strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RCstr auxotrophs was unchanged from that found in normally growing cells. In contrast, RCrel strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

scholarly journals Sublethal Concentrations of Pleurocidin-Derived Antimicrobial Peptides Inhibit Macromolecular Synthesis in Escherichia coli

2002 ◽  
Vol 46 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Aleksander Patrzykat ◽  
Carol L. Friedrich ◽  
Lijuan Zhang ◽  
Valentina Mendoza ◽  
Robert E. W. Hancock
Keyword(s):  
Escherichia Coli ◽  
Viable Count ◽  
Cationic Peptides ◽  
Macromolecular Synthesis ◽  
High Concentrations ◽  
Toxic Concentrations ◽  
Inhibition Pattern ◽  
Sublethal Concentrations ◽  
Broth Dilution ◽  
Peptide Treatment

ABSTRACT Cationic bactericidal peptides are components of natural host defenses against infections. While the mode of antibacterial action of cationic peptides remains controversial, several targets, including the cytoplasmic membrane and macromolecular synthesis, have been identified for peptides acting at high concentrations. The present study identified peptide effects at lower, near-lethal inhibitory concentrations. An amidated hybrid of the flounder pleurocidin and the frog dermaseptin (P-Der), two other pleurocidin derivatives, and pleurocidin itself were studied. At 2 μg/ml, the MIC, P-Der inhibited Escherichia coli growth in a broth dilution assay but did not cause bacterial death within 30 min, as estimated by viable count analysis. Consistent with this, P-Der demonstrated a weak ability to permeabilize membranes but was able to translocate across the lipid bilayer of unilamellar liposomes. Doses of 20 μg/ml or more reduced bacterial viable counts by about 2 log orders of magnitude within 5 min after peptide treatment. Abrupt loss of cell membrane potential, observed with a fluorescent dye, dipropylthiacarbocyanine, paralleled bacterial death but did not occur at the sublethal, inhibitory concentrations. Both lethal and sublethal concentrations of P-Der affected macromolecular synthesis within 5 min, as demonstrated by incorporation of [3H]thymidine, [3H]uridine, and [3H]histidine, but the effects were qualitatively distinct at the two concentrations. Variations of the inhibition pattern described above were observed for pleurocidin and two other derivatives. Our results indicate that peptides at their lowest inhibitory concentrations may be less capable of damaging cell membranes, while they maintain their ability to inhibit macromolecular synthesis. Better understanding of the effects of peptides acting at their MICs will contribute to the design of new peptides effective at lower, less toxic concentrations.

A new gene involved in expression of fructose-1, 6-diphosphate aldolase activity in Escherichia coli

1979 ◽  
Vol 25 (8) ◽  
pp. 937-939 ◽  
Author(s):  
Alan G. Atherly ◽  
Paul Russell
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
New Gene ◽  
Aldolase Activity ◽  
High Concentrations

A new gene, fdaB, has been mapped by transduction and partial diploid analyses and is located adjacent to argA at 59.9 min on the Escherichia coli recalibrated linkage map. This gene is involved in expression of fructose-1, 6-diphosphate aldolase activity and indirectly in ribosomal RNA synthesis. The temperature-sensitive mutant strain AA-157, containing the defective gene product of fdaB, accumulates high concentrations of fructose 1, 6-diphosphate at the nonpermissive temperature.

NUCLEIC ACID SYNTHESIS IN X-IRRADIATED THYMOCYTES

1966 ◽  
Vol 44 (6) ◽  
pp. 839-852 ◽  
Author(s):  
D. K. Myers ◽  
Kirsten Skov
Keyword(s):  
Dna Synthesis ◽  
Rna Synthesis ◽  
Acid Synthesis ◽  
X Irradiation ◽  
High Concentrations ◽  
Effects Of Radiation ◽  
Inhibition Of Dna Synthesis ◽  
Thymus Cells ◽  
Rna And Dna

The rate of incorporation of thymidine into the deoxyribonucleic acid (DNA) of rat thymocytes in vitro was not immediately affected by low doses of X-radiation, but became progressively more inhibited as the irradiated cells were incubated at 37 °C for periods of up to 6 hours. Ribonucleic acid (RNA) synthesis deteriorated almost at the same rate as DNA synthesis after X-irradiation in vitro, but protein synthesis was slightly more resistant. Addition of 10–50 mM nicotinamide to the irradiated cell suspensions tended to retard the development of this inhibition, particularly at low temperatures, but high concentrations of nicotinamide were also toxic to these cells. The inhibition of DNA synthesis by X-irradiation appeared to follow a qualitatively similar pattern in thymus, spleen, and regenerating liver in vivo.In addition to the inhibition resulting from degenerative processes in the irradiated cells, DNA synthesis in vitro was directly inhibited by 20–30 kr X-radiation. The effects of radiation on DNA synthesis paralleled to some extent its effects on the gel-forming capacity of the deoxyribonucleoprotein from the thymus cells. It is suggested that the normal synthesis of both RNA and DNA in thymocytes depends on the integrity of the deoxyribonucleoprotein.

scholarly journals spoT-dependent accumulation of guanosine tetraphosphate in response to fatty acid starvation in Escherichia coli

1993 ◽  
Vol 90 (23) ◽  
pp. 11004-11008 ◽  
Author(s):  
M Seyfzadeh ◽  
J Keener ◽  
M Nomura
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Specific Inhibitor ◽  
Acid Synthesis ◽  
Rrna Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Guanosine Tetraphosphate ◽  
A Subunit ◽  
Metabolic Signal

We previously isolated a mutant of Escherichia coli that is preferentially affected in the synthesis of rRNA and has a mutation in the gene (accD) encoding a subunit of acetyl-CoA carboxylase. Using this mutant and other mutants of the pathway for fatty acid and phospholipid biosynthesis as well as cerulenin, a specific inhibitor of fatty acid synthesis, we show that (i) inhibition of fatty acid synthesis in the presence of both a carbon source and all 20 amino acids stimulates the accumulation of guanosine tetraphosphate (ppGpp) and leads to preferential inhibition of rRNA synthesis, (ii) this ppGpp accumulation is spoT dependent, and (iii) the generation of the metabolic signal that stimulates this spoT-mediated response probably does not depend on either phospholipid starvation or a significant reduction in the level of ATP.

scholarly journals Nucleic acid synthesis and nucleotide pools in purine-deficient Escherichia coli

1970 ◽  
Vol 120 (1) ◽  
pp. 117-124 ◽  
Author(s):  
Gillian A. Thomas ◽  
N. F. Varney ◽  
K. Burton
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Rna Synthesis ◽  
Adenine Nucleotides ◽  
Acid Synthesis ◽  
Purine Nucleotides ◽  
Low Concentration ◽  
Intracellular Atp ◽  
Nucleotide Pools

1. The synthesis of nucleic acids and the content of purine nucleotides have been studied in selected purine-requiring strains of Escherichia coli including a purB− strain and a purB−guaA− strain. 2. When the exogenous purines can be converted into GTP but not into ATP, RNA is synthesized at the expense of intracellular ATP, ADP and AMP. 3. Net synthesis of RNA as measured by the incorporation of uracil can be correlated with the availability of GTP except when ATP falls to a very low concentration. 4. Nicotinamide nucleotides are not an important reservoir of adenine nucleotides for RNA synthesis.

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