Relationships between Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐Pyrophosphate in Escherichia coli

Abstract

The intracellular contents of nucleotides and 5-phosphoribosyl l-pyrophosphate (P-Rib-PP) in cells of Escherichia coli were quantitatively determined under various conditions of growth. When the growth rate was limited by the carbon source available in the growth medium the intracellular content of each of ATP, CTP, G!rP, UTP, ADP and GDP was proportional to the growth rate. The intracellular content of P-Rib-PP showed a similar, though stronger dependence, suggesting that a t the slower growth rates, the availability of P-Rib-PP could become limiting for nucleotide biosynthesis.Several treatments which are known to specifically stimulate the rate of net RNA synthesis in E. coli had a common effect in causing a transient depletion of intracellular ribonucleoside triphosphates. For each treatment, the depletion of triphosphates coincided with transiently elevated levels of P-Rib-PP, despite increases in the ADP : ATP ratio, an effect which might be expected to inhibit P-Rib-PP synthetase activity in E . coli (Atkinson and Fall [15], Klungssyr et al.[lS]). Explanations of this apparent anomaly might be made on the basis of the properties of P-Rib-PP synthetase from 8almonella typhimurium, (Switzer, [22], Switzer and Sogin, [21]). One explanation is that the enzyme is regulated by total concentrations of nucleotides so that decreases in nucleoside triphosphate concentrations result in stimulation of the reaction. Another is that the decrease in nucleoside triphosphate concentrations releases from chelation a pulse of free Ng2+ which stimulates the synthetase. And thirdly, the concentration of the substrate, ribose 5-phosphate, might be initially high enough to exert its inhibitory effect on the enzyme, so that stimulation of the reaction can occur if the substrate concentration falls.Evaluation of the regulatory mechanisms which have been proposed for control of nucleotide metabolism in. vivo in bacteria requires a knowledge of the intracellular concentrations of various substrates and possible effectors of enzyme activity, and how these concentrations vary with changes in the conditions to which the bacteria are subjected. Few reliable quantitative data of this type are currently available, and consequently, the functioning in vivo of many postulated regulatory mechanisms has little supporting evidence.We have used the thin-layer chromatographic techniques developed by Randerath and coworkers [l, 21 to

NTP-sensing by rRNA promoters in Escherichia coli is direct

“…Naturally occurring conditions in which NTP concentrations change and affect rRNA transcription have Previously, we and others (15,31) have reported that ATP and GTP concentrations increase with the increases in growth rate achieved by varying the carbon source. This correlation between steady-state growth rate and initiating NTP concentrations suggested that NTP-sensing might contribute to the phenomenon referred to as growth rate-dependent control of rRNA synthesis (15).…”

“…5A) or chloramphenicol (data not shown) to log-phase cultures, the ATP concentration increased Ϸ2-fold within 30 min relative to the ATP level in an untreated culture (see also ref. 31). The increase in ATP concentration was accompanied by an increase in transcription from rrnB P1, as measured by primer extension (Fig.…”

The Effects of Bases and Nucleosides on the Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐pyrophosphate in Escherichia coli

“…Descriptions of other materials and methods used for growth of bacteria and for estimation of intracellular nucleotides and P-Rib-PP have appeared elsewhere [3,5,6]. …”

“…Unequivocal proof of the functioning of these mechanisms may be difficult to provide, but some insight into the likelihood of their operation can be gained by measuring the intracellular concentrations of the relevant reactants, products and possible effectors and how these concentrations change with changes in conditions of growth. This approach has been previously applied to evaluating the regulation of P-Rib-PP synthetase in Escherichia coli in vivo [3].…”

Biological Insights from a Simulation Model of the Critical FtsZ Accumulation Required for Prokaryotic Cell Division

“…31 The concentration of nucleotides within the cell is known to vary with growth conditions, 32,33 Thus, as summarised in Table 1 and shown in Figure 5, at 42°C we predict a reduction in the average length of the membrane-bound FtsZ polymers. This in turn results in a dramatic lowering of χ.…”