Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Contribution to lignocellulose degradation and DOC formation from a salt marsh macrophyte by the ascomycete Phaeosphaeria spartinicola

Abstract Specifically radiolabeled [¹⁴C-lignin] lignocellulose and uniformly [U-¹⁴C] lignocellulose from the salt marsh grass Spartina alterniflora were incubated with the ascomycete Phaesphaeria s spartinicola . This fungus is the predominant one found on decaying standing dead S. alterniflora leaves in the salt marsh ecosystem. After 45 days of incubation at 20°C, 3.3% of the lignin moiety was mineralized to ¹⁴CO₂ and 2.7% solubilized to DO¹⁴C. Mineralization of the polysaccharides was seven times faster than that of the lignin. About 22% of the radioactivity was evolved as ¹⁴CO₂ but merely 4% was solubilized to DO¹⁴C within the incubation time. Experiments monitoring the ergosterol content of the mycelium incubated with S. alterniflora plant material were done to elucidate the carbon conversion efficiency of the fungus as well as the influence of the cinnamyl phenols p -coumaric and ferulic acid on lignocellulose degration. After 21 days of incubation, P. spartinicola showed a growth yield of 0.45 and 0.38 with and without the additional cinnamyl phenols, respectively. Grown on unextracted S. alterniflora the fungus caused a loss of organic plant material of about 50% with a corresponding growth yield of 0.38 during the incubation period. Investigation of cupric oxide oxidation products of sound and degraded lignocellulose revealed a preferential utilization of the syrinyl and cinnamly phenols compared with vanilly phenols.     

Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

Abstract The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like ³H-thymidine ¹⁴C-uracil, ¹⁴C-leucine and ¹⁴C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.     

The fate of carbon during the assimilation of carbamoyl phosphate in white spruce seedlings as revealed by [14C]-carbamoyl phosphate, [14C]-cyanate, and [14C] -bicarbonate labeling patterns

The fate of carbamoyl phosphate in white spruce seedlings revolves around the production of spontaneous degradation products, cyanate, bicarbonate, and carba-mate. When [¹⁴C]-carbamoyl phosphate and [¹⁴C]-cyanate are assimilated, urea is a common early metabolic intermediate that appears in the alcohol soluble N. By contrast, urea is not detected among the products of [¹⁴C]-bicarbonate. Carbamoyl phosphate and glutamic acid are implicated as having pivotal roles in the production of amides, arginine, and biotin. Within 2-h exposures to radioactive substrates considerably more carbon from bicarbonate was diverted into amino acids Incorporated into proteins than with carbon-nitrogen substrates. Specific activities of bound amino acid residues support the view that proteins formed from these [¹⁴C]-substrates have different rates of metabolic turnover.     

Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica

Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [³H]thymidine into bacterial DNA and sodium [¹⁴C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 10⁸ to 4.5 × 10⁹ cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [³H]thymidine and possibly also for sodium [¹⁴C]acetate.     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

Rapid labelling of bacteria with [14C]palmitic acid and its application in an aggregation assay

Abstract A rapid and simple procedure for labelling bacterial cells based on binding of [¹⁴C]palmitic acid to the bacterial surface is described. The method was found convenient for both Gram-positive and Gram-negative bacteria such as staphylococci, streptococci and Salmonella . Some factors affecting the binding of [¹⁴C]palmitic acid to the surface of streptococci were examined. Treatment of bacteria with heat, trypsin or β-galactosidase had no effect on labelling. The binding was relatively independent of ionic strength in the range 0.1–1 M NaCl, but was dependent on pH and presence of detergents. The [¹⁴C]palmitic acid labelling method was tested in studies of aggregation of oral streptococci. The aggregation assay was sensitive and very reproducible.     

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ¹³C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ¹³C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ¹³C values (> 50‰) in this microbial community. The δ¹³C of ANME-1 rods ranged from −24‰ to −87‰. The δ¹³C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ¹³C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ¹³C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ¹³C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.     

REDISTRIBUTION OF ADENINE DERIVATIVES AMONG SUBCELLULAR FRACTIONS FROM GUINEA PIG NEOCORTICAL TISSUES, ON INCUBATION IN VITRO

Abstract— After the brief in vitro exposure of guinea-pig neocortical tissue to [¹⁴C]adenine, synaptosomal fractions prepared from the incubated tissue contained about 6% of its retained ¹⁴C. On continued incubation and superfusion with or without stimulation, the synaptosomal proportion of the ¹⁴C increased, while the protein and K content of the fraction underwent smaller changes only. Colchicine, 0.5 m m , diminished the synaptosomal enrichment in [¹⁴C]adenine derivatives and also in some cases increased the ¹⁴C effluent from tissues to superfusates. Colchicine also diminished the uptake of adenosine, but not of adenine, to the neocortical tissues. It is concluded that nerve terminal regions receive adenine derivatives from other tissue components as part of their normal metabolism, and that much of this can arrive by extracellular fluids; transport cytoplasmically is not excluded.     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

EFFECT OF INSULIN ON THE INCORPORATION OF 14C OF RADIOACTIVE GLUCOSE INTO GLYCOGEN AND CARBON DIOXIDE IN CEREBRAL CORTICAL SLICES

Abstract— Slices from the cerebral cortices of normal and alloxan-diabetic rats were incubated with [U-²⁴C]glucose. When insulin was added to the incubation medium the incorporation of ¹⁴C into glycogen was significantly increased in both groups. Insulin did not appear to have any significant effect on the incorporation of ¹⁴C into carbon dioxide.     

Allocation of carbon to growth, storage and respiration in the vegetative barley plant

Abstract. It is proposed that the growing plant can be divided into three compartments with reference to carbon: soluble, storage and structural. Experiments carried out at 10, 15, 20 and 30°C in the light followed changes in size of these compartments in barley plants 10–24 days old. The redistribution of ^I4C photo-assimilated by 10 day old plants was monitored simultaneously. The soluble and storage compartments are a higher percentage of plant weight at lower temperatures, and are turned over rapidly at all temperatures; they form the source of respired ¹⁴C. About 30% of the ¹⁴C fixed enters structural material; in the first 24 h after labelling, for each unit of ¹⁴C entering the structural compartment, between 0–9 (at 15°C) and 3.2 (at 30°C) units of ¹⁴C are lost by respiration. At 15°C in the dark, respiratory loss of ¹⁴C is initially from soluble and storage compartments; thereafter respiration of ^I4C occurs at the expense of structural material.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

Benthic algae support zooplankton growth during winter in a clear-water lake

We used stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes to assess the importance of benthic algae for the zooplankton individual growth in winter in a shallow, clear subarctic lake. The δ¹³C values of calanoid ( Eudiaptomus graciloides ) and cyclopoid ( Cyclops scutifer ) zooplankton in autumn suggest a food resource of pelagic origin during the ice-free period. The zooplankton δ¹³C values were high in spring compared to autumn. E. graciloides did not grow over winter and the change in δ¹³C was attributed to a decrease in lipid content during the winter. In contrast, the increase in δ¹³C values of C. scutifer over the winter was explained by their growth on organic carbon generated by benthic algae. The δ¹⁵N of the C. scutifer food resource during winter was low compared to δ¹⁵N of the benthic community, suggesting that organic matter generated by benthic algae was mainly channelled to zooplankton via ¹⁵N-depleted heterotrophic bacteria. The results demonstrate that benthic algae can sustain zooplankton metabolic demands and growth during long winters, which, in turn, may promote zooplankton growth on pelagic resources during the summer. Such multi-chain omnivory challenges the view of zooplankton as mainly dependent on internal primary production and stresses the importance of benthic resources for the productivity of plankton food webs in shallow lakes.     

Microbial transformation of labile dissolved organic matter into humic-like matter in seawater

Abstract Microbial transformation of labile, low molecular weight dissolved organic matter (DOM) into dissolved humic matter (DHM) was studied in seawater. Surface water samples were amended with [¹⁴C into ¹⁴CO₂, TO¹⁴C (total organic ¹⁴C), and PO¹⁴C (particulate organic ¹⁴C), was measured over time in confined samples. The humic and non-humic fractions of DO¹⁴C (dissolved organic ¹⁴C) were separated according to a common operational definition of DHM based on adsorption on XAD-8 macroporous resin. Both TO¹⁴C and non-humic DO¹⁴C decreased during the experiments. However, ¹⁴C-labelled DHM increased during the first week of the incubations, to a level where it comprised 15% of the TO¹⁴C remaining in the samples, or 3% of the initially added ¹⁴C. Towards the end of experiments (ca 70 days), the humic fraction of DO¹⁴C gradually approached the background level of poisoned control samples. Provided that the XAD-8 operational definition of DHM is accepted, this study indicates that humic matter may be formed in seawater within days from labile monomers such as glucose.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.