Putrescine distribution in Escherichia coli studied in vivo by C nuclear magnetic resonance

In order to study the intracellular polyamine distribution in Escherichia coli, ¹³C-NMR spectra of [1,4-¹³C]putrescine were obtained after addition of the latter to intact bacteria. The ¹³C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using ¹³C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound [¹³C]putrescine could be displaced by addition of an excess of [¹²C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with [1,4-¹³C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements η, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T₁ values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, τ_c = 4·10⁻⁷ s for the latter. T₁ and τ_c value for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.     

Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-¹⁴C]putrescine and [1,4-¹⁴C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Polyamine synthesis in maize cell lines

Uptake of [¹⁴C]putrescine, [¹⁴C]arginine, and [¹⁴C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.     

Effects of D-glucose upon D-fuctose metabolism in rat hepatocytes: A 13C NMR study

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-¹³C]fructose, D-[2-¹³C]fructose or D-[6-¹³C]fructose (also 10 mM) in the presence of D₂O. The identification and quantification of ¹³C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of ¹³C-enriched D-fructose, decrease the production of ¹³C-enriched D-glucose, restrict the deuteration of the ¹³C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-¹³C]fructose, and to accentuate the lesser deuteration of the C₂ (as compared to C₅) of ¹³C-enriched D-glucose derived from D-[2-¹³C]fructose. The ratio between C2-deuterated and C₂-hydrogenated L-lactate, as well as the relative amounts of the CH₃-, CH₂D-, CHD₂ and CD₃- isotopomers of ¹³C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C₁ of D-[1-¹³C]glucose generated by hepatocytes exposed to D-[1-¹³C]fructose or D-[6-¹³C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-¹³C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [¹⁴C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N¹-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N¹-acetylspermidine.The close time-dependent correlation between induced putrescine formation and enhanced formation of N¹-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

An NMR study of the loss of carbon-20 in the biosynthesis of gibberellin A3 by Gibberella fujikuroi

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-¹³C] mevalonate to ¹³C-enriched C₁₉-gibberellins, plus ¹³CO₂ (derived from the loss of carbon-20). The formation of [¹³C]-gibberellins could be observed in vivo using ¹³C NMR; however that of ¹³CO₂ could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the ¹³CO₂ produced to be observed using ¹³C NMR. During incubations of H¹⁴CO₂Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.     

NMR structure analysis of uniformly 13C-labeled carbohydrates

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of ¹³C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly ¹³C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-¹³C)-sucrose, 342 Da] and one compound of medium molecular weight (¹³C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The ¹³C resonances are traced using ¹³C–¹³C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the ¹³C resonances, the ¹H chemical shifts are derived in a straightforward manner using one-bond ¹H–¹³C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the ¹ J _CC splitting of the ¹³C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either ¹³C or ¹H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T₂ relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the ¹H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the ¹³C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with ¹⁵N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and ¹³C-detected (H)CACO spectra.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil

[¹³C₆]salicylate, [U-¹³C]naphthalene, and [U-¹³C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [¹³C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the ¹³C during the incubation did not appear to be an issue in this experiment.     

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no ¹³C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated ¹³C-MFA using the optimal tracers [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-¹³C]glucose and [U-¹³C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO₂ originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH₂, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.