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.     

Lipids of Pseudomonas aeruginosa Cells Grown on Hydrocarbons and on Trypticase Soy Broth

Lipids were extracted from cells of Pseudomonas aeruginosa grown on a pure hydrocarbon (tridecane), mixed hydrocarbons (JP-4 jet fuel), and on Trypticase Soy Broth. Total lipids produced from each substrate represented from 7.1 to 8.2% of cellular dry weight, of which 5.0 to 6.4% were obtained before cellular hydrolysis (free lipids) and 1.7 to 2.0% were extracted after cellular hydrolysis (bound lipids). Free lipids from cells grown on each medium were separated into four fractions by thin-layer chromatography. All fractions were present in cells from each type of medium, and the "neutral fraction" constituted the largest fraction. The fatty acid composition of free lipids was determined by gas-liquid chromatography. Cells grown on each medium contained saturated and unsaturated C(14) to C(20) fatty acids. Trace amounts of C(13) fatty acids were found in tridecane-grown cells. Saturated C(16) and C(18) were the major acids present in all cells. Quantitative differences were found in fatty acids produced on the three media, but specific correlations between substrate carbon sources and fatty acid content of cells were not evident. Tridecane-grown cells contained only traces of C(13) acid and small amounts of C(15) and C(17) acids, suggesting that the organism's fatty acids were derived from de novo synthesis rather than by direct incorporation of the hydrocarbon.     

Role of 4-aminobutyrate aminotransferase in the arginine metabolism of Pseudomonas aeruginosa.

4-Aminobutyrate aminotransferase (GABAT) from Pseudomonas aeruginosa was purified 64-fold to apparent electrophoretic homogeneity from cells grown with 4-aminobutyrate as the only source of carbon and nitrogen. Purified GABAT catalyzed the transamination of 4-aminobutyrate, N2-acetyl-L-ornithine, L-ornithine, putrescine, L-lysine, and cadaverine with 2-oxoglutarate (listed in order of decreasing activity). The enzyme is induced in cells grown on 4-guanidinobutyrate, 4-aminobutyrate, or putrescine as the only carbon and nitrogen source. Cells grown on arginine or on glutamate contained low levels of the enzyme. The regulation of the synthesis of GABAT as well as the properties of the mutant with an inactive N2-acetyl-L-ornithin 5-aminotransferase suggest that GABAT functions in the biosynthesis of arginine by convertine N2-acetyl-L-glutamate 5-semialdehyde to N2-acetyl-Lornithine as well as in catabolic reactions during growth on putrescine or 4-guanidinobutyrate but not during growth on arginine.     

Turnover of Putrescine in a Pseudomonas Species

A Pseudomonas species was found to readily take up labeled putrescine added in trace amounts to any of four growth media, bis-(3-aminopropyl)-amine, 4-aminobutyrate, glucose-NH(3), and Casamino Acids, although the rate of uptake varied considerably from one medium to another. Putrescine degradation, as well as excretion and conversion to hydroxyputrescine, was demonstrated in all four media, indicating that this organism has a constitutive putrescine degradation pathway. The extents of putrescine degradation, excretion, and conversion to hydroxyputrescine are shown for these four growth media through an incubation period of 1 hr. These results document more fully the experimental details behind a previous communication which postulated that the constitutive degradation of putrescine participates in the regulation of intracellular putrescine concentration. The significance of this apparent violation of the general concept that synthetic end products are normally not degraded is discussed.     

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.     

Studies on the endogenous metabolism and senescence of starved Sarcina lutea

1. When washed suspensions of Sarcina lutea are starved aerobically in phosphate buffer at the growth temperature of 37 degrees , the rate of endogenous oxygen consumption decreases to very low values after 10hr., although many of the cells survive for 40hr. If starvation is prolonged further, the bacteria die at a rate of approximately 1.5% of the initial viable population per hour. 2. Oxidation of intracellular free amino acids accounts for most of the observed endogenous oxygen uptake but RNA is also utilized and a portion of the component bases and pentose is degraded and presumably oxidized. Ammonia appears in the supernatant and some pentose and ultraviolet-absorbing nucleotide are released from the cells. DNA, protein and polysaccharide are not measurably degraded. 3. Survival can be correlated with the ability of aerobically starved bacteria to oxidize exogenous l-glutamate and glucose. When starved under nitrogen for 40hr. cells continue to oxidize their endogenous reserves at undiminished rates when transferred to aerobic conditions; on prolonging anaerobic starvation the rate of oxidation declines during the period of most rapid loss of viability. 4. In the presence of Mg(2+), RNA degradation during aerobic starvation is almost completely suppressed without affecting the period for which the bacteria survive. 5. Cells grown in peptone supplemented with glucose accumulate reserves of polysaccharide which are metabolized in aerobic starvation, together with free amino acids. Ammonia is evolved and RNA is degraded to a greater extent than in peptone-grown suspensions. Bacteria rich in polysaccharide survive less well than those which are deficient in the polymer; the reason for this phenomenon has yet to be established. 6. In peptone medium, endogenous oxygen uptake and the concentration of intracellular free amino acids decline as growth progresses and they continue to decrease when the organism is held in stationary phase. Under the conditions used, the endogenous Q(o2) and free amino acid pool of cells grown in peptone with 2% (w/v) glucose did not decline so markedly and the bacteria contained large amounts of polysaccharide at all stages of growth.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes

Asparagine stimulated the translation of ornithine decarboxylase (ODC) mRNA more than 10-fold in cultured hepatocytes which had been pretreated with glucagon in simple salt/glucose medium. Putrescine suppressed the increase in the rate of ODC synthesis caused by asparagine without significant change in the amount of ODC mRNA, suggesting that putrescine inhibited the effect of asparagine at least in part at the level of translation. Polysomal distribution of ODC mRNA was analyzed to examine the site of translational regulation by these effectors. In uninduced hepatocytes, most of the ODC mRNA was sedimented slightly after the 40 S ribosomal subunit. This ODC mRNA was sequestered from translational machinery since it was not shifted to the polysome fraction when peptide elongation was specifically inhibited by a low concentration of cycloheximide. In asparagine-treated cells, 40% of total ODC mRNA was in the polysomal fraction and formed heavier polysomes, indicating that asparagine stimulated both recruitment of ODC mRNA from the untranslatable pool and the initiation steps of translation. Putrescine did not change the distribution pattern of ODC mRNA on polysomes significantly. Thus, 30% of ODC mRNA remained on polysomes even when ODC synthesis was completely inhibited by putrescine. Paradoxically more than 70% of ODC mRNA was shifted into polysomes by putrescine in the presence of low concentrations of cycloheximide. These results, together with changes in the polysome profile, suggested that putrescine nonspecifically stimulated the recruitment of ODC mRNA from the untranslatable pool, whereas it specifically inhibited its translation at both the initiation and the elongation steps.     

Putrescine uptake by the cellular slime mould dictyostelium discoideum.

1. Rapid labelling occurs when myxamoebae of Dictyostelium discoideum strain AX2 are incubated with [1,4-14C]putrescine. Labelling is energy-dependent. 2. The label enters a pool from which rapid exchange with extracellular putrescine does not occur, and labelling is believed to represent uptake into the cells. 3. The concentration-dependence of putrescine uptake indicates that a number of systems are involved, at least one of which is saturable, with a Km of 9.1 micro M-putrescine. At high putrescine concentrations the overall uptake process is non-saturable. 4. Significant metabolism of putrescine and loss of intracellular putrescine to the medium only occurred when cells were incubated with millimolar concentrations of extracellular putrescine. 5. Putrescine uptake was inhibited by diamines, polyamines, bivalent metal ions and omega-aminocarboxylic acids. 6. The ability to take up putrescine at low concentrations decreased during starvation of myxamoebae. 7. The results are interpreted in terms of a model for putrescine uptake involving adsorptive pinocytosis at low concentrations and fluid-phase pinocytosis at high concentrations.     

Polyamines and protein synthesis: studies in various polyamine-requiring mutants of Escherichia coli.

Different Escherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. The in vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutans MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.     

Multiple Transport Components for Putrescine in Escherichia coli

Putrescine uptake was studied in cultures of Escherichia coli K-12 grown in media of high or low osmolarity. When grown in high osmolarity medium, a transport system of low K(m) and low V(max) was found. For cultures grown in a medium of low osmolarity, the kinetics of putrescine uptake was more complex and consistent with the existence of an additional transport system of higher K(m) and V(max). This conclusion is supported by the isolation of mutants in which one or the other system appears to be defective and by the ability of chloramphenicol to block the expression of the second transport system. Both systems appear to prefer putrescine over other compounds, since several basic amino acids and other polyamines competed only weakly for transport. The action of both uptake systems was shown to cause significant displacement of intracellular putrescine. Both systems also are at least partially energy dependent.     

The nature of the proteins present in the 'relaxed particles' from methionine-starved Escherichia coli A19 (Hfr rel met rns).

The 'relaxed particles' formed during methionine starvation of Escherichia coli A19 (Hfr rel met rns) have been isolated by large-scale rate-zonal density gradient ultracentrifugation. The proteins and rRNA species associated with these particles have been examined. The rRNA species present are precursor and mature forms of 16S and 23S rRNA. The bulk of the rRNA which accumulates during starvation is found within the particles. The proteins prepared directly from the particles give strong multiple immunoprecipitates with antisera specific to 30S and 50S ribosomal proteins. The soluble proteins, prepared and examined in the same manner, do not give this immunological reaction. Two-dimensional electrophoresis patterns of the proteins from the particles show that the proteins co-migrate with proteins from 30S and 50S ribosomes and are entirely dissimilar to the proteins prepared by the same methods from the soluble fraction of the cells. On the basis of these and other observations, it is concluded that the 'relaxed particles' are not artefacts but are arrested ribosome precursors containing both rRNA and certain ribosomal proteins. The free pool of ribosomal proteins is low in exponential-phase cells and is not significantly increased by a 2 h period of starvation for glucose. The implications of these observations concerning the proteins associated with 'relaxed' and 'chloramphenicol particles' are discussed in raltion to ribosome biogenesis and the stabilization of rRNA.     

Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.     

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.     

Physiological Studies in Plant Nutrition: XVIII. Some Aspects of Nitrogen Metabolism in Barley and other Plants in Relation to Potassium Deficiency

Barley was grown in water and sand culture using a variety ofnutrient solutions, the most important variable being potassiumsupply. The diamine putrescine, which is not normally foundin this plant in appreciable amounts, is present in the potassium-deficientseedling and accumulates during its growth. Maximum accumulationcoincides with the development of the severe symptoms associatedwith advanced deficiency. Putrescine occurs at an earlier stagein potassium-deficient plants whose nitrate and phosphate aresupplied as ammonium salts than in those to which the correspondingcalcium salts are given. It is found less abundantly in theroots than in the tops. It is absent from protein hydrolysates. A leaf necrosis characteristic of potassium-deficiency is inducedby feeding putrescine to barley having a high potassium status;after prolonged feeding the appearance of such plants suggestssevere potassium starvation. The putrescine is slowly utilized,and at the same time an unidentified substance is produced.What seems to be the same substance appears in quantity in high-potassiumred clover during the course of rapid utilization of administeredputrescine. It also occurs naturally in potassium-deficientred clover. When potassium is supplied to potassium-deficientbarley in which putrescine has accumulated, the amine soon disappears.Application of either rubidium or sodium also leads to a reductionin putrescine, though to a lesser extent, rubidium being themore effective element. Under conditions of extreme potassium-deficiency wheat and redclover also accumulate putrescine. The free amino-acid compositionof the wheat then bears a striking resemblance to that of deficientbarley.     

A possible role of histidine in a nickel resistant mechanism of Saccharomyces cerevisiae

When a nickel resistant strain N08 of S. cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction. The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine. When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation. The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.     

The selection of cultured plant cell lines producing high levels of biotin

We have found that biotin is synthesized in many species of cultured plant cells, e.g. Lavandula vera Labiatae), Nicotiana tabacum (Solanaceae) and Glycine max Leguminosae). Cultured green L. vera cells grown under light contained the greatest amounts of free biotin of the cells studied although the specific amounts varied among the cell lines. Cell lines were selected after their free biotin contents had been analysed. Cells containing large amounts of free biotin were cultured repeatedly, analysed and reselected. Lines with high levels of free biotin were obtained from cells which survived on a medium containing pimelic acid and l-alanine or from gamma irradiated cells. One L. vera cell line obtained from irradiated cells contained seven times the amount of free biotin found in the original unselected cultured cells and four and a half times that found in the leaves.     

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE,NITRITE, AND AMMONIA BY BIDDULPHIA AURITA1

Biddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source. Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non-protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time. The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble-free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium. Cells grown in the nitrate medium have both nitrate- and nitrite-reductase activity. Cells grown in the nitrite medium have only nitrite-reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.     

Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.