Heterotrophic Carbon Metabolism by Beggiatoa alba

The assimilation and metabolism of CO(2) and acetate by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(2) for growth, the addition of excess CO(2) (as NaHCO(3)) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to (14)CO(2) by B. alba. The apparent V(max) values for combined assimilation and oxidation of [2-(14)C]acetate by B. alba were 126 to 202 nmol min(-1) mg of protein(-1) under differing growth conditions. The V(max) values for CO(2) assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min(-1) mg of protein(-1), respectively. The low V(max) values for CO(2) assimilation, coupled with the high V(max) values for acetate oxidation, suggested that the required CO(2) was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by B. alba for the fixation of CO(2). From 61 to 73% of the [(14)C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-(14)C]acetate was incorporated into poly-beta-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-beta-hydroxybutyric acid. Succinate and CO(2) were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-beta-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-(14)C]acetate assimilation. Glutamate and aspartate were the first stable (14)CO(2) fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable (14)CO(2) fixation products over a 30-min period. The CO(2) fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO(2) fixation mechanisms are absent from B. alba B18LD and that the CO(2) and acetate metabolism pathways are probably linked.     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.     

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.     

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.     

The Substrate Utilization and Concentration of 14C Photosynthates in Citronella under Fe Deficiency

Changes in the utilization pattern of primary substrate, viz. [U-14C] acetate, 14CO2 and [U-14C] saccharose, and the contents of 14C fixation products in photosynthetic metabolites (sugars, amino acids, and organic acids) were determined in Fe-deficient citronella in relation to the essential oil accumulation. There was an overall decrease in photosynthetic efficiency of the Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essential oil after 14CO2 had been supplied. When acetate and saccharose were fed to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and organic acids, there was a lower incorporation of these metabolites into essential oils than in control plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation, severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citronella.     

Chloroflexus aurantiacus secretes 3-hydroxypropionate,a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 M). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO₂ fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using ¹³C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO₂. The 3HP formed by cells grown on propionate and ¹³CO₂ was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO₂ fixation.Abbreviations 3HP 3-hydroxypropionate - 2F3HP 2,fluoro,3-hydroxypropionate - FAc fluoroacetate - GC gas chromatography - MS mass spectrometry - NMR nuclear magnetic resonance     

A transfer of carbon atoms from fatty acids to sugars and amino acids in yellow lupine (Lupinus luteus L.) seedlings

The metabolism of 14C-acetate was investigated during the in vitro germination of yellow lupine seeds. Carbon atoms (14C) from the C-2 position of acetate were incorporated mainly into amino acids: aspartate, glutamate, and glutamine and into sugars: glucose, sucrose, and fructose. In contrast to this, 14C from the C-1 position of acetate was released mainly as 14CO2. Incorporation of 1-14C and 2-14C from acetate into amino acids and sugars in seedling axes was more intense when sucrose was added to the medium. However, in cotyledons where lipids are converted to carbohydrates, this process was inhibited by exogenous sucrose. Since acetate is the product of fatty acid beta-oxidation, our results indicate that, at least in lupine, seed storage lipids can be converted not only to sucrose, but mainly to amino acids. Inhibitory effects of sucrose on the incorporation of 14C from acetate into amino acids and sugars in cotyledons of lupine seedlings may be explained as the effect of regulation of the glyoxylate cycle by sugars.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Acetate Assimilation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. It has been found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate shunt. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 313–318.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Tsygankov, Laurinavichene, Ivanovsky.     

Carbon assimilation pathways in sulfate reducing bacteria. Formate,carbon dioxide,carbon monoxide,and acetate assimilation by Desulfovibrio baarsii

Desulfovibrio baarsii is a sulfate reducing bacterium, which can grown on formate plus sulfate as sole energy source and formate and CO₂ as sole carbon sources. It is shown by ¹⁴C labelling studies that more than 60% of the cell carbon is derived from CO₂ and the rest from formate. The cells thus grow autotrophically. Labelling studies with [¹⁴C]acetate, ¹⁴CO and [¹⁴C]formate indicate that CO₂ fixation does not proceed via the Calvin cycle. The labelling patterns of alanine, aspartate, glutamate, and glucosamine indicate that acetate (or activated acetic acid) is an early intermediate in formate and CO₂ assimilation; the methyl group of acetate is derived from formate, and the carboxyl group from CO₂ via CO; pyruvate is formed from acetyl-CoA by reductive carboxylation. The capacity to synthesize an acetate unit from two C₁-compounds obviously distinguishes D. baarsii from those Desulfovibrio species, which require acetate as a carbon source in addition to CO₂.     

Respiration of an obligate methylotroph in the presence of various substrates]

Respiration of the cells of Methylococcus ucrainicus, strain 21, cultivated in the atmosphere of methane, is stimulated by methanol, formaldehyde, formate, n-alcohols, and allyl alcohol. The rate of oxygen assimilation is lower in the presence of isopropanol, isobutanol, propane, butane, maltose, and some organic acids (acetate, fumarate, citrate, succinate). The Michaelis constant for methanol is 88 mcM. Oxidation of methane, methanol, formaldehyde, and formate by the bacterium is inhibited by cyanide, hydroxylamine, and azide. The rate of oxygen assimilation by the cells in the presence of methane and other C1-compounds did not decrease after the suspension had been stored at 4 degrees C during four months and longer.     

Short-term Products of 14C-acetate Assimilation by Chlorella pyrenoidosa in the Light

The kinetics of ¹⁴C-2-acetate assimilation by Chlorella pyrenoidosain the light were examined. Under aerobic conditions the primaryproduct of acetate assimilation was succinic acid which, afterten seconds, contained over 60 per cent of the ¹⁴C incorporatedby the cells. The percentage of the total ¹⁴C in succinate fellwith time, while that in citrate and glutamate increased. After1800 sec over 60 per cent of ¹⁴C was present in two compounds,glutamic acid and an unknown compound (X). Glucose-6-phosphate,fructose-6-phosphate, phosphoglyceric acid and phosphoenolpyruvicacid became labelled after 60 sec but together never containedmore than one per cent of the total ¹⁴C incorporated. Underanaerobic conditions succinate was still the primary productof acetate assimilation, and the absence of carbon dioxide resultedin a decrease in ¹⁴C incorporation into compound X. The patternof acetate assimilation in acetate grown and acetate adaptedChlorella was very similar to that in photo-autotrophicallygrown Chlorella. In the presence of 10^–6M DCMU, succinicacid was the primary product of acetate assimilation, but therewas an early Incorporation of ¹⁴C into glutamate, aspartate,and malate. 4 x10^–3M MFA did not effect the early incorporationof ¹⁴C into succinic acid, but resulted in accumulation of ¹⁴Cin citrate and a decreased amount in glutamate and in compound X.     

Inorganic Carbon-Stimulated O2 Photoreduction Is Suppressed by NO2- Assimilation in Air-Grown Cells of Synechococcus UTEX 625

The effect of NO2- assimilation on O2 exchange and CO2 fixation of the cyanobacterium, Synechococcus UTEX 625, was studied mass spectrometrically. Upon addition of 1 mM inorganic carbon to the medium, inorganic carbon pools developed and accelerated O2 photoreduction 5-fold when CO2 fixation was inhibited. During steady-state photosynthesis at saturating light, O2 uptake represented 32% of O2 evolution and balanced that portion of O2 evolution that could not be accounted for by CO2 fixation. Under these conditions, NO2- assimilation reduced O2 uptake by 59% but had no influence on CO2 fixation. NO2- assimilation decreased both CO2 fixation and O2 photoreduction at low light and and increased net O2 evolution at all light intensities. The increase in net O2 evolution observed during simultaneous assimilation of carbon and nitrogen over carbon alone was due to a suppression of O2 photoreduction by NO2- assimilation. When CO2 fixation was precluded, NO2- assimilation inhibited O2 photoreduction and stimulated O2 evolution. When the electron supply was limiting (low light), competition among O2, CO2, and NO2- for electrons could be observed, but when the electron supply was not limiting (saturating light), O2 photoreduction and/or NO2- reduction caused electron transport that was additive to that for maximum CO2 fixation.     

13C-NMR study of autotrophic CO2 fixation in Thermoproteus neutrophilus

The pathway of autotrophic CO2 fixation has been investigated in the extremely thermophilic sulfur-respiring anaerobic archaebacterium Thermoproteus neutrophilus. [1,4-13C2]Succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from CO2. Three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several generations in the presence of [1,4-13C2]succinate and their labelling patterns were determined by 13C NMR spectroscopy. The data is consistent with CO2 fixation by a reductive citric acid cycle, as proposed earlier for the green sulfur bacterium Chlorobium limicola, the sulfate-reducing Desulfobacter hydrogenophilus and the microaerophilic Knallgasbacterium Hydrogenobacter thermophilus. The presence of a reductive citric acid cycle in archaebacteria indicates that this CO2 fixation mechanism which is an alternative to the Calvin cycle is present in many anaerobic or facultative anaerobic microorganisms.     

Glutamate racemization and catabolism in Fusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.     

Differences of heterotrophic 13CO2 assimilation by Pseudomonas knackmussii strain B13 and Rhodococcus opacus 1CP and potential impact on biomarker stable isotope probing

Motivated by the finding that Pseudomonas knackmussii B13 but not Rhodococcus opacus 1CP grows in the absence of externally provided CO(2), we investigated the assimilation of (13)CO(2) into active cells cultivated with non-labelled glucose as sole energy substrate. (13)C found in the bulk biomass indicated a substantial but different CO(2) assimilation by Pseudomonas and Rhodococcus, respectively (3500 per thousand and 2600 per thousand). Cellular fatty acids were labelled from -15 per thousand to 470 per thousand and amino acids from 500 per thousand to 24,000 per thousand demonstrating clear differences between various compound classes. 'You are what you eat plus 1 per thousand' is therefore only valid for the average bulk C without specific isotope signature deviation of the external CO(2) or carbonates. Odd-numbered and 10-methyl fatty acids, which are much more abundant in Rhodococcus or other Gram-positive bacteria, were up to fivefold higher enriched in (13)C relative to the Pseudomonas fatty acids. A high-level growth-phase-independent, labelling of the oxaloacetate-derived amino acids indicated heterotrophic CO(2) fixation by anaplerotic reactions known to replenish the tricarboxylic acid cycle. Although both strains assimilate CO(2) via similar general pathways, Rhodococcus depends to a much higher extent on carboxylations reactions with external CO(2) owing to the formation of odd-numbered fatty acids. As a general consequence, heterotrophic fixation of CO(2) should be taken into account in investigations of degradation experiments using isotope tracer compounds.     

Exogenous Glutamate Is Metabolized to Glutamine and Exported by Rat Primary Astrocyte Cultures

Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous L-[U-14C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid-precipitated cell fraction. More than 84% of the label was recovered in these fraction. suggesting that the maximum possible formation and loss of 14CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol X mg protein-1 X min-1 when 9 microM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.