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F C Neidhardt

Publications and source records attributed to F C Neidhardt.

At least 73 records · Page 4Linked to original sources
PubMed1978

Effect of inhibitors of elongation factor Tu on the metabolic regulation of protein synthesis in Escherichia coli.

By using inhibitors of elongation factor Tu (L-1-tosylamido-2-phenylethyl chloromethyl ketone [TPCK] and kirromycin), we determined the effect of elongation factor Tu inhibition on the synthesis of individual components of the translation machinery. The rates of synthesis of individual proteins were measured in double-label experiments using a two-dimensional gel system. TPCK inhibition produce a coordinate decrease in the differential synthesis rates of all components of the translation machinery examined in these experiments. On the other hand, kirromycin inhibition increased the differential synthesis rates of some translation components and decreased the differential synthesis rates of others. These results suggest that the metabolic regulation of synthesis of various translation proteins is not mediated through a common signal.

Amino Acid Chloromethyl Ketones↗
PubMed1978

Regulation of lac operon expression: reappraisal of the theory of catabolite repression.

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.

Culture Media↗
PubMed1977

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli.

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.

Alanine-tRNA Ligase↗
PubMed1977

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli.

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.

Acetates↗
PubMed1977

Physiological regulation of a decontrolled lac operon.

The expression of the lac operon was studied under a variety of growth conditions in induced and in constitutive cells of Escherichia coli that carried different catabolite-insensitive lac promoters. Use of such "decontrolled" lac operons permitted a study of the expression of an operon that was presumably subject only to passive control. Since the use of toluenized cells was demonstrated not to be completely reliable, all enzyme assays were performed on sonic supernatant fluids. The cells contained different catabolite-insensitive promoters, which included the L1 and UV5 lac promoters, as well as others isolated in this study. There were three major observations. First, small but real carbon source effects were seen. Second, there was only a small change in beta-galactosidase specific activity with changes in the growth rate. This result implies a limited transcription and/or translation capacity within the cell. Third, at rapid growth rates, most promoters exhibited a decreased expression. The UV5 promoter, which was the "strongest" promoter, was an exception. A mechanism to explain this promoter-dependent control is discussed.

Acetylesterase↗
PubMed1976

The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli A.

The effects of a partial restriction of valyl-tRNA aminoacylation on the synthesis of aminoacyl-tRNA synthetases, ribosomal proteins, and other translation and transcription proteins were examined in otherwise isogenic stringent (relA+) and relaxed (relA1) derivatives of E. coli B. The synthesis of individual ribosomal proteins, elongation factor G, and to a lesser extent elongation factors Tu and Ts, and the valyl- and arginyl-tRNA synthetases was found to be subject to the influence of the stringent control system. The synthesis of the alpha and beta subunits of RNA polymerase and several of the aminoacyl-tRNA synthetases, in contrast, is either not subject to the influence of the stringent control system, or is subject to additional regulatory constraints.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Growth rate modulation of four aminoacyl-transfer ribonucleic acid synthetases in enteric bacteria.

The specific activities of arginyl- glutamyl- seryl-, and valyl-transfer ribonucleic acid (tRNA) synthetases were measured in the wild-type and mutant strains of Salmonella typhimurium LT2 and Escherichia coli B/r. In media restricted only by carbon and energy source availability, the specific activities of all four enzymes were proportional to the growth rate, with the exception of seryl-tRNA synthetase in S. typhimurium, which remained essentially constant. Structural gene densities were calculated for these four enzymes and were found not to account for the variation of specific activity with growth rate.

Amino Acids↗
PubMed1975

Physiological and biochemical studies on the function of 5-methyluridine in the transfer ribonucleic acid of Escherichia coli.

Matched pairs of transductant strains differing by the presence of absence of 5-methyluridine (ribothymidine) (m5U) in their transfer ribonucleic acid (tRNA) were used to study the function of this modified nucleoside in Escherichia coli. Ordinary measurements of growth rate in different media revealed no effect of the loss of m5U in tRNA. A gene located close to trmA (the structural cistron for the methyltransferase that produces m5U in tRNA), however, was found to reduce the growth rates significantly, depending on the medium and the temperature of cultivation. Measurement of codon recognition, macromolecular composition, tRNA binding to the ribosome, and the rate of protein chain elongation in vivo indicated no disadvantage caused by the lack of m5U. The regulation of ilv and his operons seemed also to be unaffected by the absence of m5U in the tRNA. In a mixed population experiment, however, cells possessing m5U in their tRNA seemed to have a distinct advantage over cells lacking this modified nucleoside. This experiment provides the first indication of the overall value of m5U in tRNA.

Escherichia coli↗
PubMed1974

Culture medium for enterobacteria.

A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

Ammonia↗
PubMed1972

Unusual valyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.

Adenosine Triphosphate↗
PubMed1972

Growth-linked instability of a mutant valyl-transfer ribonucleic acid synthetase in Escherichia coli.

The valyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli strain NP2907, previously described as having an elevated K(m) for adenosine triphosphate and reduced stability in vitro compared to the wild type, was found to be conditionally thermolabile in vivo. The rate of inactivation of this enzyme at a particular temperature appears to be coordinated with the rate of growth; at 40 C this coordination results in equal rates of synthesis and destruction over a wide range of growth rates. In vitro studies showed that conditions favoring maintenance of the valyl-tRNA synthetase-valyl adenylate complex conferred complete protection against inactivation at 40 C, whereas the further addition of uncharged tRNA caused rapid, irreversible decay. We propose that the rate of inactivation of this mutant valyl-tRNA synthetase in vivo is a function of the ratio of deacylated to acylated tRNA(val) and that this ratio is a function of growth rate. The event which renders the valyl-tRNA synthetase susceptible to inactivation is likely to be the normal breakdown of the valyl-tRNA synthetase-valyl-adenylate complex during a cycle of aminoacylation of tRNA(val).

Acylation↗