Polyamine Synthesis and Accumulation in Escherichia coli Infected with Bacteriophage R17

We have studied the biosynthesis of polyamines during the multiplication of the RNA bacteriophage R17. R17-sensitive strains of Escherichia coli were derived from the stringent CP78 and the relaxed mutant derivative CP79. The cells were infected with R17 in the presence or absence of arginine, a required amino acid, and both the RNA and polyamine contents of the bacteria were determined before and after the infection. The uninfected CP79 rel derivative accumulated RNA and spermidine in the absence of arginine, unlike the stringent organism that accumulated neither under these conditions. After R17 infection, the stringent strain accumulated RNA and spermidine in the presence or absence of arginine. The data indicate a close correlation between the synthesis of RNA and spermidine, suggesting a significant role for this polyamine in the multiplication of phage R17.     

Polyamines and the Accumulation of Ribonucleic Acid in Some Polyauxotrophic Strains of Escherichia coli

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA.Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.     

Biochemical mechanism of uracil uptake regulation in Escherichia coli B. Allosteric effects on uracil phosphoribosyltransferase under stringent conditions.

The regulation of uracil uptake in bacteria was studied in bacteriophage T4-infected cells, where host-specific, stable RNA synthesis is completely shut-off by phage, and where phage-specific RNA synthesis, which is not stringently regulated, could be followed by a continuous incorporation of uracil. This incorporation into phage RNA was found to be dependent on the allelic state of the rel gene and it was thus severely restricted under stringent conditions. This was not the case with adenine, which was incorported into RNA to almost the same extent under stringent and relaxed conditions, respectively. The inhibition of uracil uptake under proceeding RNA formation, which was furthermore found to be reversed by addition of chloramphenicol, indicated a specific mechanism governing the cellular entry of uracil. This is suggested to involve the allosteric regulation of uracil phosphoribosyltransferase (EC 2.4.2.9.). The enzyme was partially purified by ammonium sulfate precipitation and gel chromatography. The dependence on GDP and GTP as positive effectors was demonstrated. The stimulatory effect of GTP was abolished in vitro by the addition of guanosine 5'-diphosphate 3-diphosphate, which is known to accumulate during amino acid starvation in stringent bacteria. The reversible inactivation of the enzyme by dilution suggested a subunit structure of uracil phosphoribosyltransferase.     

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.     

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid II. Requirement for Polyamines in T4 Infection of a Polyamine Auxotroph

Polyamine depletion produced by exogenous arginine in Escherichia coliK-12 cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral pH results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as N-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine.     

Isolation of Escherichia coli mutants with changed regulation of uracil uptake.

The incorporation of uracil into the pyrimidine ribonucleotide pools of Escherichia coli is strongly restricted under stringent conditions. Previously, we have suggested that this inhibition can be explained by the allosteric properties of uracil phosphoribosyltransferase. It has been proposed that this enzyme performs the uptake of uracil into the cell by transporting it across the cytoplasmic membrane, with the stimultaenous formation of UMP. To test this hypothesis it would be helpful to have mutants with changed regulation of uracil uptake, and in the present work, a method is introduced for the selection of such mutants. This method is based on phenotypic suppression of amber mutations by 5-fluorouracil (5FU). Mutants were isolated in an arginine-requiring strain of E. coli carrying an amber mutation in argI, the ornithine transcarbamylase gene. To facilitate the phenotypic rescue of this defective gene, mutants which overproduced ornithine transcarbamylase mRNA were isolated as a first step. The absence of exogenously added arginine causes stringent conditions, and phenotypic rescue by 5FU is, thus, prevented, unless the 5FU uptake mechanism is mutationally changed in such a manner that the drug is taken up into the cell. Three mutants in which the growth could be supported by 5FU in the absence of arginine were isolated. Two of them had acquired an increased ability to take up uracil under stringent conditions.     

Isoelectric focusing of fibrinogens on polyacrylamide gels

The thiol-oxidizing agent, diamide, has been used to convert glutathione to glutathione disulfide within the cells of a stringent strain of Escherichia coli (CP 78), leading to a cessation of 14C-leucine incorporation (protein synthesis) and 3H-uracil incorporation (RNA synthesis). Parallel experiments with an isogenic relaxed strain (CP 79) gave similar results, providing evidence that glutathione is closely linked to RNA synthesis indepently of the link previously shown to protein synthesis.     

Effect of 9-beta-D-arabinofuranosyladenine on polymer synthesis in a polyauxotrophic strain of Escherichia coli

Leung, Hazel Barner (University of Pennsylvania School of Medicine, Philadelphia), Alice McGovern Doering, and Seymour S. Cohen. Effect of 9-beta-d-arabinofuranosyladenine on polymer synthesis in a polyauxotrophic strain of Escherichia coli. J. Bacteriol. 92:558-564. 1966.-Adenine-requiring mutants have been obtained from Escherichia coli strain 15 TAU, which also needs thymine, arginine, and uracil for growth. Some of these are killed by 9-beta-d-arabinofuranosyladenine (ara-A) in the absence of exogenous adenine; a particular mutant of this type, designated TAUAd, has been used in our studies. The lethality of ara-A, d-arabinosylhypoxanthine, and the 1-n-oxide of ara-A has been compared; ara-A is equally toxic in the presence or absence of thymine. Although the absence of uracil reduces ara-A toxicity, the lack of arginine almost eliminates lethality. It was found that ara-A completely inhibits deoxyribonucleic acid synthesis without markedly affecting ribonucleic acid (RNA) synthesis. Some inhibition of protein synthesis can be detected. However, the interpretation of these results is complicated because (i) exogenous adenine must be excluded, (ii) endogenous adenine is made available from RNA turnover, and (iii) ara-A is being rapidly converted to only slightly less toxic arabinosylhypoxanthine by the adenosine deaminase of E. coli. A suitable inhibitor for the bacterial deaminase has not yet been found.     

Polyamines in bacteriophage R17 and its RNA.

Bacteriophage R17 and its RNA were found to contain significant amounts of spermidine but not of putrescine. When isolated at 0.01 M KCl, up to 1,000 molecules of spermidine were associated with the virion. The phage RNA isolated with phenol plus sodium lauryl sulfate contained approximately 70 to 90 molecules of spermidine. The association appeared to be ionic because the bound spermidine could be dissociated by KCl, MgCl2, or both. Effects of polyamines on in vitro translation were studied using both poly(U) and phage R17-RNA as mRNA. Addition of spermidine to the system at suboptimal concentrations of Mg2+ resulted in marked stimulations of the rate of protein synthesis. Putrescine alone had no effect but stimulated the incorporation in the presence of suboptimal concentrations of spermidine plus Mg2+. The isolated amino acid-incorporating system contained suboptimal soluble and bound polyamines. A comparison of incorporation was made in this system using R17-RNA with and without bound spermidine. No effects of these bound cations were detected on the rate or extent of incorporation of valine. The ratio of incorporation of histidine (present in non-coat proteins) to valine (total protein) revealed little difference as a functions of cation in the system or a function of the spermidine present in R17-RNA.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in chloramphenicol-inhibited cultures of Escherichia coli

The rate of polymerization of ribosomal ribonucleic acid chains was estimated for steadily growing cultures of Escherichia coli M.R.E.600, from the kinetics of incorporation of exogenous [5-³H]uracil into completed 23S rRNA molecules. The analytical method of Avery & Midgley (1971) was used. Measurements were made at 37°C, in the presence or the absence of chloramphenicol, in each of three media; enriched broth, glucose–salts or sodium lactate–salts. The rate of chain elongation of 23S rRNA was virtually constant in all media at 37°C, as 24±4 nucleotides added/s. Accelerations in the rate of biosynthesis of rRNA by chloramphenicol in growth-limiting media are due primarily to an increase in the rate of initiation of new RNA chains, up to the rates existing in cultures growing rapidly in broth. Thus, in poorer media, only a small fraction of the available DNA-dependent RNA polymerase molecules are active at any given instant, since the chain-initiation rate is limiting in these conditions. In cultures growing rapidly in enriched broth, antibiotic inhibition caused a rise of some 12% in the rate of incorporation of exogenous uracil into total RNA. This small acceleration was due entirely to the partial stabilization of the mRNA fraction, which accumulated as 14% of the RNA formed after the addition of chloramphenicol. In cultures growing more slowly in glucose–salts or lactate–salts media, chloramphenicol caused an immediate acceleration of two- to three-fold in the overall rate of RNA synthesis. Studies by DNA–RNA hybridization showed that the synthesis of mRNA was accelerated in harmony with the other affected species. However, just over half the mRNA formed after the addition of chloramphenicol quickly decayed to acid-soluble products, whereas the remainder was more stable and accumulated in the cells. The mRNA fraction constituted about 6% of the total cellular RNA after 3h inhibition. A model was suggested to explain the partial stabilization and accumulation of the mRNA fraction and the acceleration in the rate of synthesis of mRNA when chloramphenicol was added to cultures in growth-limiting media.     

Effect of 5-fluorouracil on the growth of bacteriophage R17

Graham, A. F. (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and Clare Kirk. Effect of 5-fluorouracil on the growth of bacteriophage R17. J. Bacteriol. 90:928-935. 1965.-When added to Escherichia coli within 2 min after phage R17, 5-fluorouracil (FU), at a concentration of 10(-4)m, completely inhibited the synthesis of infectious ribonucleic acid (RNA) and phage. If the addition of FU was made later than 5 min after infection, infectious RNA synthesis was blocked but infectious phage was still formed; the infectious RNA made before the addition of FU continued to be incorporated into mature phage. These properties of the inhibitor were used to determine the kinetics of phage RNA synthesis and the size of the phage precursor RNA pool. At a concentration of 2.2 x 10(-5)m FU, the yield of phage was reduced to 15% of that in an uninhibited control, 28% of the phage RNA uracil was replaced with FU, and the specific infectivity of the phage was unaltered.     

Stable Ribonucleic Acid Synthesis in Stringent (rel+) and Relaxed (rel−) Polyamine Auxotrophs of Escherichia coli K-12

The relationship of polyamines to stable ribonucleic acid (RNA) synthesis under conditions of amino acid withdrawal or chloramphenicol treatment was examined with the use of a closely related rel(+), rel(-) pair conditionally incapable of synthesizing putrescine. Under conditions of polyamine starvation, the cellular sperimidine level fell to one-third to one-half of the value observed in putrescine-supplemented cultures and putrescine became undetectable; cadaverine was synthesized by both strains, but the relaxed strain, MA 252, accumulated less cadaverine per cell than its stringent twin, MA 254. Upon amino acid withdrawal, the stringent strain remained stringent whether starved of or supplemented with polyamines. Similarly, the relaxed strain was capable of making RNA either with or without polyamine starvation. On the addition of chloramphenicol or upon amino acid withdrawal in the relaxed strain, supplementation with spermidine had no effect on the initial rate of RNA synthesis, although RNA accumulation was greater in the presence of added spermidine. Spermidine added at the conclusion of RNA synthesis prompted additional synthesis, although preincubation with spermidine again had no effect on the initial rate. All forms of stable RNA species were made with polyamine supplementation. The present data appear to rule out the possibility that polyamines are primary causative agents in stimulating RNA synthesis, but rather suggest an indirect or secondary role for spermidine in which the polyamines "stimulate" stable RNA synthesis probably by relieving RNA product inhibition of RNA synthesis.     

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

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.     

Spermidine Biosynthesis During Germination and Subsequent Vegetative Growth of Bacillus megaterium Spores

Spermidine biosynthesis was extremely low early in germination of Bacillus megaterium spores and the spermidine level remained constant. Rapid synthesis began after 130 min and thereafter accounted for the increase in spermidine level which began at this time. Biosynthesis was greatly (>84%) diminished by exogenous spermine or spermidine. Arginine and ornithine were both converted efficiently into spermidine, but arginine was the more immediate precursor as shown by isotope competition studies and by the absence of ornithine decarboxylase and the presence of arginine decarboxylase. Exogenous putrescine was not incorporated into spermidine, although it was taken up rapidly and degraded.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.