Nutritional Shift-up Research Articles

Modifications of membrane lipid composition following the nutritional shift-up of starved cells a comparison with membrane biogenesis in Tetrahymena

Detailed analyses of lipid composition have been made on various membrane fractions isolated at different intervals after 24 h-starved Tetrahymena cells were refed with nutrient-rich medium. During starvation there was a marked alteration in both phospholipid polar headgroup and acyl chain compositions: an increase in 2-aminoethylphospholipid and γ-linolenic acid (18 : 3) with a concurrent decrease in phosphatidylethanolamine and palmitoleic acid (16 : 1). However, following refeeding, such an altered lipid composition was rather rapidly restored to the initial level of the control cell membranes prior to starvation. This membrane lipid modification was found to occur in good accordance with the recovery of cell size and lipid synthesis. The considerable changes in the principal unsaturated fatty acids, 16 : 1 and 18 : 3, which are formed via the palmitate and stearate desaturation pathways, respectively, were suggested to be accounted for by the levels of desaturases activities. The results of the labeling experiments with radioactive precursors have demonstrated that in the refed cells, there was a more rapid and dynamic transfer or exchange between membranes as compared with that in the exponentially growing control cells. Thus, rapid ameliorative modifications of membrane lipid composition are thought to be required for the urgent growth of membrane systems in the refed cell which should be ready to initiate new division.

Dimensional rearrangement of rod-shaped bacteria following nutritional shift-up. II. Experiments with Escherichia coli[formula omitted

The dimensions of Escherichia coli B r (strain H266) in transition between two states of balanced growth, were determined from electron micrographs of fixed cells by sampling the culture at various times following nutritional shift-up from a doubling time of 72 min to one of 24 min. Mean cell length rises immediately and overshoots its final steady-state value, cell diameter increases monotonically; both approach their asymptotic levels only after several hours. The results are compared with the dimensions predicted by each of two models of cell growth and morphogenesis in rod-shaped bacteria. The first attributes cell elongation to circular zones that double in number at a particular time during the cell cycle and which act at rates proportional to the growth rate; the second is similar, except that it considers surface growth rather than length extension as the active process, length being determined passively. Two possibilities are examined, that the zonal growth rate adjusts immediately to the new growth conditions, and that it does so gradually. The experimental data appear consistent with the gradual response version of the surface growth model.

Dimensional rearrangement of rod-shaped bacteria following nutritional shift-up. I. Theory

Theoretical expressions are derived for two models that describe average length and radius of a population of rod-shaped bacteria as a function of time following their transfer to a medium that supports a higher growth rate. The first attributes cell elongation to circular zones produced at a particular time during the cell cycle and which act thereafter at rates proportional to the growth rate; the second is formally identical but considers surface growth rather than length extension. Two possibilities are considered, that the zonal growth rate adjusts immediately to the transition, and that it does so gradually. The results are also displayed graphically, covering a broad range of each of the various parameters involved; values are chosen to permit a direct comparison between the models. Average cell length is seen to undergo a large overshoot and to approach its steady-state value from above, while cell radius remains almost constant or even decreases somewhat before increasing monotonically towards its asymptotic level; both require a considerable period of time to reach steady state. The transient behavior predicted by the two models is found to be quite different even when the steady-state dimensions are identical; the differences between immediate and gradual response of the zonal growth rate are even greater. It is shown that using a dimensionless measure of cell geometry, the aspect ratio, can facilitate selection of the appropriate model.

Synthesis and function of ribonucleic acid polymerase and ribosomes in Escherichia coli B/r after a nutritional shift-up.

The syntheses of stable ribosomal ribonucleic acid (RNA) and transfer RNA in bacteria depend on the concentration and activity of RNA polymerase and on the fraction of active RNA polymerase synthesizing stable RNA. These parameters were measured in Escherichia coli B/r after a nutritional shift-up from succinate-minimal to glucose-amino acids medium and were found to change in complex patterns during a 1- to 2-h period after the shift-up before reaching a final steady-state level characteristic for the postshift growth medium. The combined effect of these changes was an immediate, one-step increase in the exponential rate of stable RNA synthesis and thus of ribosome synthesis. This suggests that the distribution of transcribing RNA polymerase over ribosomal and nonribosomal genes and the polymerase activity are continuously adjusted during postshift growth to some growth-limiting reaction whose rate increases exponentially. It is proposed that this reaction is the production of amino-acylated transfer RNA and that is exponentially increasing rate results in part from a gradually increasing concentration of aminoacyl transfer RNA synthetases after a shift-up. This idea was tested and is supported by a computer simulation of a nutritional shift-up.

Synthesis of ribosomal protein S1 following nutritional shift-up in Escherichia coli K-12.

The synthesis rate of ribosomal protein S1 was measured in Escherichia coli K-12 during the transitional period following a nutritional shift-up from acetate minimal to glucose/amino acids/nucleosides medium. The synthesis rate of S1 increased without a lag suggesting that the S1 gene is under stringent control and located very close to its promoter. The rate of S1 synthesis slowed between 7 and 15 min, and then increased to the postshift-up steady state rate. The postshift-up steady state rate was half the initial rate obtained between 0 and 7 min. The slow down in the synthesis rate between 7 and 15 min indicates that an unknown factor(s), in addition to guanosine 5'-diphosphate, 3'-diphosphate, participates in the regulation of S1 gene expression.

In vivo regulation of chromosomal beta-lactamase in Escherichia coli

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.

Cell division during nutritional upshifts of Escherichia coli

Nutritional shifts of Escherichia coli B/r to richer media have been analyzed is synchronously growing and exponential-hase populations. Early perturbations in the timing of cell division were observed. At the slow growth, division progressed at a rate equal to or less than the preshift rate for about 1 h. At intermediate growth, both delays and acceleration in division were observed. The extent of the perturbation depended upon the age of the cells at the time of the shift and the composition of the preshift and postshift media. The perturbation was different in the two substrains of E. coli B/r used in this study.

Acetate kinase production by Escherichia coli during steady-state and transient growth in continuous culture

The synthesis of acetate kinase by Escherichia coli ATCC 9637 was studied during growth in anaerobic continuous cultures under steady-state and transient conditions. During growth in anaerobic, glucose-limited chemostats, acetate kinase synthesis was linearly associated with growth. Two types of non-steady-state transients were studied: the perturbation in one was the addition of glucose alone, and, in the second, glucose plus Casamino Acids. During the nutritional shift-up in the second case, but not in the first, the instantaneous specific acetate kinase activities and specific synthesis rates exceeded pre- and postshift values. Trajectory curves demonstrated that the increase in specific activity remained within the bounds of values obtainable under steady-state conditions with minimal and Casamino Acids media. Specific synthesis rates, however, greatly exceeded steady-state values. Enzyme yield values on glucose after the transient nutritional shift-up increased up to fivefold. Active protein synthesis is shown to be necessary to achieve the enhanced specific synthesis rates and enzyme yields. The results from these transient responses are discussed in terms of a conceptful model for metabolic regulation.

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the ββ′ subunits of RNA polymerase following a nutritional shiftup of Escherichia coli

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.

Synthesis of messenger RNA coding for individual ribosomal proteins during nutritional shift-up in Escherichia coli K-12

The rates of increase in mRNA content coding for the individual ribosomal proteins were measured in Escherichia coli K-12 following a nutritional shift-up. Messenger RNA synthesis was arrested by the inhibitor thiolutin and the amount of protein synthesized from the pre-existing mRNA was used as a measure of mRNA content. The data obtained show, with the exception of protein S2, a rapid increase in the mRNA content during the first 7 min after shift-up followed by a slower rate of increase during the period 7–30 min. The amount of protein S2-specific mRNA increased at a rate that remained constant over the entire 0–30 min period. Among the remaining ribosomal protein cistrons a variety of rates of transcription was obtained during both the early and later periods; these rates were not related to the amount of mRNA present immediately prior to shift-up. The significance of the anomalous behaviour of protein S2 is discussed as well as the change in rate of mRNA synthesis observed for the remainder of the ribosomal proteins at about 7 min following shift-up. The results are discussed in relation to other data on the regulation of ribosomal protein synthesis and to the grouping of cistrons into transcriptional units.

Polyamine levels in Escherichia coli during nutritional shiftup and exponential growth

At different exponential growth rates obtained either by varying the carbon source of the culture medium or limiting glucose uptake, intracellular levels of putrescine and spermidine were measured. Over a ten-fold increase in growth rate an approximately three-fold increase in putrescine level and a 3.5-fold increase in spermidine level per cell absorbance were observed. Conditions favoring an abrupt alteration in growth rate, such as occur following nutritional shiftup of Escherichia coli, resulted in a significant increase in the intracellular level of putrescine and virtually no change in the spermidine level. Because of the magnitude and the timing of the change in polyamine levels, the hypothesis that polyamines are (the components) responsible for inducing the rapid increase in the rate of RNA synthesis following nutritional shiftup is rejected.

Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein

The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.

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.

Synthesis of Ribosomal and Transfer Ribonucleic Acids in Yeast During a Nutritional Shift-up

The growth rate of Saccharomyces cerevisiae was increased by adding a mixture of amino acids to cultures containing proline as the sole nitrogen source. The transition from balanced growth in the basal medium (doubling time 4 h) to balanced growth in the enriched medium (doubling time 2 h) took about 2-5 h. The rate of RNA accumulation increased soon after the enrichment to almost its final value. This increase began after a short lag of 10 to 15 min, therefore synthesis of new RNA polymerase molecules may be required before stable RNA production can increase. The different stable RNA species were not stimulated at different times after the enrichment, but all increased continuosly throughout the transition. The rRNA species accumulated in a co-ordinate fashion at a rate faster than the rate of tRNA accumulation.

Kinetics of ribosome synthesis during a nutritional shift-up inEscherichia coli K-12

The rates of total protein synthesis, polyribosome formation and 70S ribosome accumulation were measured following a nutritional shift-up of Escherichia coli K-12. Changes in ribosome content and distribution during the shift-up were measured by examining the total cellular content of free and polysome-associated ribosomes using a sensitive double isotope labeling method. The kinetics of ribosomal subunit formation and the biosynthesis of subunit protein and RNA species were also defined. The results indicated that a pre-shift population of ribosomal subunits was utilized for the immediate post shift increase in both total and ribosomal-specific protein synthesis. An assembly time for new subunits of about 3 min was observed. The formation of certain ribosomal proteins during the shift suggested that new subunit assembly was limited by the rate of synthesis of particular ribosomal proteins during this growth transition.

Growth response of Escherichia coli to nutritional shift-up: immediate division stimulation in slow-growing cells.

When Escherichia coli 15T- cells growing exponentially at 70- to 80-min doubling times are subjected to a nutritional shift-up via glucose addition, cell division continues at the preshift rate for about 70 min (rate maintenance). The same cells growing at doubling times of 120 min or longer, however, begin to divide at a new faster rate immediately upon glucose addition. In both the rate maintenance and immediate division situations, cell mass, as measured by optical density (OD), begins to increase immediately upon shift-up. Consequently, the OD/cell pattern differs in the two growth-rate transitions. During rate maintenance, the OD/cell ratio increases dramatically for 60 to 70 min, and then slows appreciably and approaches the OD/cell characteristic of the new medium. During immediate division situations, the OD/cell increases only slightly for the first 180 +/- min; then the rate of increase accelerates but does not stop at the OD/cell characteristic of the new medium. Immediate division upon nutritional shift-up apparently depends upon initial doubling times in excess of 115 to 120 min and provision of a readily metabolized carbon source supporting doubling times of about 40 min. Similar immediate division occurs in E. coli B/r and K-12.

Synthesis of messenger RNA coding for elongation factors G and Ts during nutritional shift-up in [formula omitted][formula omitted] K-12

Following a nutritional shift-up, the rate of ribosomal protein synthesis increased almost immediately, whereas the rates of synthesis of elongation factors G and Ts increased after a lag of 5 – 10 minutes. The messenger RNA content during shift-up was examined using a technique involving labelling of the protein synthesized following inhibition of RNA synthesis. Messenger coding for the ribosomal proteins increased immediately, whereas that coding for the elongation factors G and Ts increased only after a lag of about 10 minutes and closely followed the pattern of messenger coding for total soluble proteins. These data suggest that the observed differences in rates of synthesis of the ribosomal and elongation factor proteins during shift-up are due to differences in the tiems of transcription of the messengers. The possibility of translational discrimination is discussed.

Evidence of the involvement of an outer membrane protein in DNA initiation.

Protein D of molecular weight 80,000 is incorporated into the membrane of Escherichia coli B/r for a brief fraction of the cell cycle. The protein is incorporated into the outer membrane; its incorporation seems to reflect de novo synthesis and, once incorporated, it is stable. In synchronous acetate-grown cultures a 10-fold increase in the rate of protein D synthesis occurs toward the end of the cell cycle, near the end of one round of DNA synthesis and the start of the next. In synchronous glucose-grown cultures the protein appears approximately halfway through the cell cycle, shortly before DNA synthesis begins. The observed correlation between protein D synthesis and DNA initiation, approximately 15 min later, is strengthened by experiments with inhibitors and mutants. Inhibition of protein synthesis at any time during DNA synthesis delays the subsequent appearance of protein D and the initiation of the next round of DNA synthesis. The synthesis of both protein D and DNA is prevented by nalidixic acid; after its removal a sharp spike of protein D synthesis is followed by a wave of DNA synthesis. Experiments with DNA and DNAB mutants, with amino acid starvation of an auxotroph and with nutritional shift-up, all supported the coupling of protein D synthesis with the initiation of DNA synthesis. The addition of beta-lactam antibiotics which affect murein metabolism dramatically increases the amount of protein D. The cells are prevented from terminating protein D synthesis; different antibiotics have different kinetics of action. Protein D preferentially binds to double-stranded DNA in vitro. We suggest that this protein provides a metabolic link between murein synthesis, protein synthesis, and DNA initiation, and that it acts at a specific time in the cell cycle as an attachment site for DNA to the cell envelope.

The relationships among RNA synthesis, RNA polymerase synthesis and guanosine tetraphosphate levels in Escherichia coli during nutritional shift-up

The relationships among the rate of RNA synthesis, RNA polymerase synthesis and activity, and guanosine tetraphosphate levels were investigated following nutritional shift-up in Escherichia coli. RNA synthesis continues at the preshift rate for 1.5 min after which an increase is observed that reaches a new steady-state rate at between 2 and 2.5 min. RNA polymerase activity measured in crude extracts increases immediately and by 10 min has increased 50%. RNA polymerase synthesis as measured by the synthesis of the β and β′ subunits lags for 2.5 min and then increases 75% by 10 min. Guanosine tetraphosphate levels decrease 50% by 3 min to levels characteristic of steady-state post-shift-up cells. The significance of these data to the regulation of RNA synthesis during shift-up is discussed.

Macromolecular synthesis after a nutritional shift-up of Bacillus subtilis.

Effects of amino acids of macromolecular synthesis in Bacillus subtilis were studied. Two mutants, CRK4001 and NIG45, that were selected as slow growers in rich media were proved to be useful to analyse early events occurring after addition of amino acids to exponentially growing cells in a glucose-salts medium (nutritional shift-up). In a wild type strain, the rate of stable RNA (sRNA: essentially ribosomal RNA) synthesis increased 2.3 fold shortly after the shift-up to the rate characteristic of the post-shift steady state growth. In contrast,sRNA synthesis in the mutant strains responded to the shift-up in two steps. Thus, shortly after the shift the rate of sRNA synthesis increased 2.2 fold as in the wild type but this increased level was maintained temporarily for 60 min and suddenly decreased to the post-shift steady state rate (1.4 fold increase). On the other hand, rates of DNA synthesis increased some 30 min after the shift directly to the post-shift steady state rates in all strains. Ratios of an origin to a terminus marker (purA/metB) began to increase exponentially to reach maximum values at 60 min after the shift, indicating that initiation of DNA replication occurred at frequencies characteristic of respective post-shift growth rates soon after the shift. These results revealed that the initial increase in the rate of sRNA synthesis and the frequency of initation of DNA replication after nutritional shift are not related to each other and are independently affected by amino acids. In concert with these findings, the increase in sRNA synthesis was not affected by inhibition of DNA synthesis for the first 60 min after the shift, while it was completely prevented by puromycin and choramphenicol. Protein synthesis for 10 min after the shift was sufficient to fully change the sRNA synthesis rate by amino acids.