Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis and Methods for Their Measurement

The rates of synthesis of Aerobacter aerogenes nucleic acids were estimated during incubation of the bacteria in a Mg(++)-free medium. Deoxyribonucleic acid (DNA) synthesized during Mg(++) starvation, or in the preceding exponential growth, remained acid-precipitable for 2.5 hr before breaking down to acid-soluble products during a period of many hours. Rates of DNA synthesis were calculated by correcting the net amounts of DNA per milliliter to values that would have appeared had there been no decay. After the first few hours, this rate was constant, the amount of DNA present at the start of Mg(++) starvation being synthesized every 130 min. Rates of synthesis of total ribonucleic acid (RNA) were established in two ways: (i) by measurements of the incorporation of exogeneous uracil and glucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA), since this component is stable during Mg(++) starvation. After the first few hours, this rate was constant, the amount of RNA present at the start of Mg(++) starvation being synthesized about every 120 min. Fractionation by gradient centrifugation revealed that at all times of starvation the ratio of newly synthesized tRNA-rRNA was the same as it was during exponential growth. Furthermore, newly synthesized ribosomal RNA (rRNA) became a part of polysomal structures. Thus, in the absence of Mg(++), DNA, tRNA, and rRNA were synthesized in the same relative proportions as during exponential growth, at rates close to one-half the instantaneous rates of synthesis in the bacteria growing exponentially at the start of starvation.     

Magnesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition

Aerobacter aerogenes incubated in a medium containing all factors necessary for exponential growth except Mg⁺⁺ continued to synthesize nucleic acids and proteins for more than 70 hr, provided the major carbon source was in excess at all times. After 24 hr of Mg⁺⁺ starvation, deoxyribonucleic acid content in the culture had increased 10-fold. In contrast, the viable-cell count increased only about threefold during the first few hours and then remained approximately constant for the subsequent 70 hr. After specified intervals of Mg⁺⁺ starvation, extracts of the bacteria, or ribonucleic acid (RNA) purified from them, was centrifuged through gradients of sucrose to separate transfer RNA from ribosomal components. After correcting for losses, we obtained the following results. (i) There was a progressive rise in the content of transfer RNA competent to accept amino acids and during starvation it remained completely stable. (ii) In contrast, the contents of normally sedimenting ribosomal RNA and ribosomal subunits (30 and 50S) remained approximately constant for more than 24 hr. This did not result from stability of ribosomes made prior to starvation together with an inhibition of synthesis of new particles. Rather, ribosomes were continually breaking down and being replaced by an equivalent number of new ones. (iii) The breakdown of ribosomes appeared to be sequentially ordered; polysomes yielded 70S monomers, which then gave 30 and 50S particles, and these disintegrated to smaller units and finally to acid-soluble products. (iv) Furthermore, the particles derived from breakdown do not appear to exchange with subparticles on the path of assembly. Thus, ribosome decay was age-dependent and ribosomal RNA molecules had a minimal life expectancy of 90 min; however, some survived much longer.     

The synthesis of ribosomes by a mutant of Escherichia coli

1. When the methionine-requiring mutant 58–161 of Escherichia coli was starved of methionine, ribonucleic acid was made in the absence of protein synthesis. 2. Most of this ribonucleic acid was similar to that found in ribosomes but was contained in particles differing from ribosomes both in sedimentation coefficient and in chromatographic behaviour on diethylaminoethylcellulose. 3. When methionine was added to a starved culture, the ribonucleic acid synthesized during starvation was almost completely undegraded as growth resumed. A transient loss of 5–10% could be largely attributed to breakdown of messenger ribonucleic acid accumulated during starvation. 4. After the addition of methionine, ribosomes were formed from the particles, and during this period preferential synthesis of ribosomal protein took place. 5. It is suggested that under these conditions the direct synthesis of ribosomes from the particles may occur.     

Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.     

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.     

Ribonucleic acid and ribosomes of Bacillus stearothermophilus

Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332-339. 1966.-The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10(-2)m MgCl(2)-10(-2)m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg(++) concentration to 10(-3)m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10(-2)m Mg(++) to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a T(m) at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a T(m) of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Regulation of Ribonucleic Acid Synthesis by Histidine and Methionine During Recovery of Escherichia coli from Magnesium Starvation

During magnesium starvation of Escherichia coli B, most of the ribosomes break down to low-molecular-weight components. When magnesium is restored to the medium, the cells recover. The rate of recovery can be increased greatly by supplementing the growth medium with a mixture of 21 amino acids. This increased rate of recovery is shown to be due to the effect of only two amino acids, histidine and methionine, which initially stimulate accumulation of cellular ribonucleic acid without increasing the rate of protein synthesis. In contrast, histidine and methionine supplementation to logarithmically growing E. coli B is not as effective in stimulating growth as is the complete amino acid mixture. Since cells recovering from magnesium starvation preferentially synthesize ribosomes, it is possible that histidine and methionine play a special role(s) in ribosomal ribonucleic acid synthesis or stability.     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

Changes in ribosome function induced by protein kinase associated with ribosomes of Streptomyces collinus producing kirromycin.

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.     

Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements.

1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo.     

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).     

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.     

Changes in the Plasma Membrane of Escherichia coli During Magnesium Starvation

The effect of Mg(++) starvation on the structure of the Escherichia coli cell membrane was studied with the freeze-etch technique. Special attention was paid to changes within the plane of the membrane, which in normal exponentially growing cells has a netlike arrangement of particles 2 to 6 nm in diameter. During Mg(++) starvation, a paracrystalline particle pattern appeared on the plasma membrane, and large areas devoid of particles were seen. Although these changes are reproducibly associated with Mg(++) starvation of the bacteria, no decrease in the Mg(++) content of the cell envelope per se was detected, even after 24 hr of Mg(++) deprivation. The structural changes caused by Mg(++) deprivation appeared to involve specific and permanent alterations in membrane development. The absence of other nutrients or divalent cations did not induce similar alterations.     

The extraction of proteins from eukaryotic ribosomes and ribosomal subunits

Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.