De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.     

Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

Alteration of De Novo Glucose Production Contributes to Fasting Hypoglycaemia in Fyn Deficient Mice

Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of ¹³C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.     

Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes.     

De Novo Purine Biosynthesis in Intact Cells of Cucurbita pepo

The capacity of intact cells of roots excised from summer squash plants (Cucurbita pepo L. cv Early Prolific Straightneck) to synthesize purine nucleotides de novo was investigated. Evidence that purine nucleotides are synthesized de novo included: (a) demonstration of the incorporation of [1-¹⁴C]glycine, [2-¹⁴C]glycine, NaH¹⁴CO₃, and H¹⁴COONa into total adenine nucleotides; (b) observation that the addition of azaserine or aminopterin, known inhibitors of de novo purine synthesis in other organisms, blocked the incorporation of these precursors into adenine nucleotides; and (c) demonstration that the purine ring synthesized from these precursors was labeled in a manner consistent with the pathway for de novo purine biosynthesis found in microorganisms and animal tissues. Under optimal conditions, the activity of this pathway in roots excised from 2-day-old squash plants was 244 ± 13 nanomoles (mean ± standard error, n = 17) NaH¹⁴CO₃ incorporated into ∑Ade (the sum of the adenine nucleotides, nucleoside and free base) per gram tissue during the 3-hour incubation period.

The possible occurrence of alternative enzymic reactions for the first steps of de novo purine biosynthesis was also investigated. No conclusive evidence was obtained to support the operation of alternative enzymic reactions in the intact cell of C. pepo.

     

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrB1), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip(+) episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB(+) is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein.     

Growth Dynamics of Salmonella enterica Strains on Alfalfa Sprouts and in Waste Seed Irrigation Water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

De Novo Biosynthesis of Volatiles Induced by Insect Herbivory in Cotton Plants

In response to insect feeding on the leaves, cotton (Gossypium hirsutum L.) plants release elevated levels of volatiles, which can serve as a chemical signal that attracts natural enemies of the herbivore to the damaged plant. Pulse-labeling experiments with [13C]CO2 demonstrated that many of the volatiles released, including the acyclic terpenes (E,E)-[alpha]-farnesene, (E)-[beta]-farnesene, (E)-[beta]-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, and (E/E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, as well as the shikimate pathway product indole, are biosynthesized de novo following insect damage. However, other volatile constituents, including several cyclic terpenes, butyrates, and green leaf volatiles of the lipoxygenase pathway are released from storage or synthesized from stored intermediates. Analysis of volatiles from artificially damaged plants, with and without beet armyworm (Spodoptera exigua Hubner) oral secretions exogenously applied to the leaves, as well as volatiles from beet armyworm-damaged and -undamaged control plants, demonstrated that the application of caterpillar oral secretions increased both the production and release of several volatiles that are synthesized de novo in response to insect feeding. These results establish that the plant plays an active and dynamic role in mediating the interaction between herbivores and natural enemies of herbivores.     

Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log₁₀ on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log₁₀. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway''s end-product. Palmitate''s major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in particular virion assembly, relies on the synthesis and mitochondrial import of fatty acids, where their β-oxidation drives robust ATP production.     

Developmental Changes in the Folate-Dependent Enzymes of De Novo Purine Biosynthesis in Rat Brain

Abstract: The activities of the two folate-dependent enzymes in the de nova purine biosynthetic pathway (e.g., glycinamide ribonucleotide transformylase and aminoimidazolecarboxamide ribonucleotide transformylase), have been evaluated as a function of age in crude extracts from rat brain, liver, kidney, and spleen. The activities of the enzymes in brain are similar to those found in liver and kidney. In all tissues the activity of both enzymes was higher during early development, more than nine times above adult levels. In the CNS the enzymatic activities are apparently related to the periods of increased nucleic acid synthesis, with different activities being found in different regions during development. Our findings lend strong support to the suggestion that folic acid-dependent metabolism plays an important role during early development of the brain.     

De Novo Biosynthesis of Nicotinamide Adenine Dinucleotide in Escherichia coli: Excretion of Quinolinic Acid by Mutants Lacking Quinolinate Phosphoribosyl Transferase

The excretion of quinolinic acid was studied in growing and resting cells of Escherichia coli K-12 nadC(13). Under optimal conditions, this organism could synthesize quinolinic acid in several-fold excess of the amount which would be required for normal growth. The excretion of quinolinic acid was controlled by the concentration of nicotinamide adenine dinucleotide (NAD) precursors available to the organism either during growth or during incubation in dense cell suspensions. These observations suggest that biosynthesis of NAD de novo is regulated by both repression and feedback inhibition. Analogues of niacin which inhibit bacterial growth also inhibited and repressed the synthesis (excretion) of quinolinic acid. The pH optimum for quinolinic acid excretion agreed favorably with the optimum observed for its synthesis in vitro. The rate of quinolinic acid excretion was strongly influenced by the concentration of ribose or glycerol in the medium.     

Amino-4-Imidazolecarboxamide Ribotide Directly Inhibits Coenzyme A Biosynthesis in Salmonella enterica

Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a by-product of histidine biosynthesis. In bacteria, yeast, and humans, accumulation of AICAR has been shown to affect an array of cellular processes by both direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when coenzyme A (CoA) levels are reduced. Our results show that the in vivo accumulation of AICAR decreased total CoA pools and, further, that AICAR inhibited the activity of pantoate β-alanine ligase in vitro (PanC, EC 6.3.2.1). These results demonstrated a mechanism of AICAR action and provide new insights into the metabolic consequences of disrupting purine metabolism.