Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

Biosynthesis of 4-methyl-1-nonanol: Female-produced sex pheromone of the yellow mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

The objective of this study was to elucidate the biosynthetic route to 4-methyl-1-nonanol, the female-produced sex pheromone of the yellow mealworm beetle, Tenebrio molitor L. The biosynthetic route to the pheromone was examined by (i) allowing the females to feed on defatted bran coated with a stable isotope-labeled putative precursor ([1-¹³C]acetate, [1-¹³C]propionate, [1-¹³C]pentanoate, [1-¹³C]2-methylheptanoic acid, or [²H₂]4-methylnonanoic acid); (ii) determining if the precursors were incorporated by analyzing the emitted pheromone by gas chromatography/selected ion monitoring-mass spectroscopy (GC/SIM-MS); (iii) where the pheromone was isotopically-enriched, determining the position of the isotopic label(s) through comparison of the MS fragmentation pattern with that of unlabelled 4-methyl-1-nonanol. Although the incorporation of [1-¹³C]acetate into 4-methyl-1-nonanol could not be detected, relatively large proportions of the pheromone were produced from the other precursors tested: 81% from [²H₂]4-methylnonanoic acid, 45% from [1-¹³C]2-methylheptanoic acid, 16% from [1-¹³C]pentanoate, and 35% from [1-¹³C]propionate (27% from only one unit, and 7.8% from two units). The results indicate that 4-methyl-1-nonanol is produced through a modification of normal fatty acid biosynthesis: initiation of the pathway with one unit of propionate results in the uneven number of carbons in the chain; incorporation of another unit of propionate during elongation provides the methyl branch; reduction of 4-methylnonanoic acid produces the alcohol pheromone. The elucidation of the biosynthetic pathway of 4-methyl-1-nonanol biosynthesis in the yellow mealworm is the first step towards understanding the biochemistry of sex pheromone production in this species.     

Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

The Assignment of the 13C-NMR Spectrum of Lysocellin and Its Biosynthesis

The complete ¹⁸C-NMR assignment of lysocellin sodium salt has been obtained based on the ¹³C-¹³C double labeling method and comparison with its retroaldol degradation product as well as a structurally related polyether antibiotic, lasalocid A.

In parallel, the biosynthesis of lysocellin was investigated by feeding ¹³C-labeled precursors followed by analyzing the resulting labeling patterns of the ¹³C-NMR spectra; a pathway to the antibiotic molecule is suggested which proceeds through condensation of two butyrate, eight propionate and one acetate units.

In addition, a conversion of butyrate to propionate during the metabolic process has been disclosed.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Photoheterotrophic Metabolism of Acrylamide by a Newly Isolated Strain of Rhodopseudomonas palustris

Acrylamide, a neurotoxin and suspected carcinogen, is produced by industrial processes and during the heating of foods. In this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions (doubling time of 10 to 12 h) but poorly under anaerobic dark or aerobic conditions. Acrylamide was rapidly deamidated to acrylate by strain Ac1, and the subsequent degradation of acrylate was the rate-limiting reaction in cell growth. Acrylamide metabolism by succinate-grown cultures occurred only after a lag period, and the induction of acrylamide-degrading activity was prevented by the presence of protein or RNA synthesis inhibitors. ¹³C nuclear magnetic resonance studies of [1,2,3-¹³C]acrylamide metabolism by actively growing cultures confirmed the rapid conversion of acrylamide to acrylate but failed to detect any subsequent intermediates of acrylate degradation. Using concentrated cell suspensions containing natural abundance succinate as an additional carbon source, [¹³C]acrylate consumption occurred with the production and then degradation of [¹³C]propionate. Although R. palustris strain Ac1 grew well and with comparable doubling times for each of acrylamide, acrylate, and propionate, R. palustris strain CGA009 was incapable of significant acrylamide- or acrylate-dependent growth over the same time course, but grew comparably with propionate. These results provide the first demonstration of anaerobic photoheterotrophic bacterial acrylamide catabolism and provide evidence for a new pathway for acrylate catabolism involving propionate as an intermediate.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

Further studies on the biosynthesis of the avermectins

Summary The biosynthesis of avermectins was studied further inStreptomyces avermitilis MA5502 by feeding experiments with labeled precursors.¹³C-NMR analysis of the compounds biosynthesized from [2-¹³C]acetate, [1,2-¹³C₂]acetate, [3-¹³C]propionate and [2,3-¹³C₂]propionate confirmed that the aglycone of avermectins is made from seven intact acetate and five propionate units. Feeding experiments with [1-¹³C]2-methylbutyrate and [1-¹³C]isobutyrate have shown that 2-methylbutyrate and isobutyrate are immediate precursors of the starter units of the polyketide chains of avermectin a and b components, respectively. The³H/¹⁴C doublelabeling experiments suggest that the two oleandrose moieties are derived from glucose.     

13C-NMR Study of propionate metabolism by sludges from bioreactors treating sulfate and sulfide rich wastewater

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution¹³ C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented.¹³ C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-¹³C]propionate into [2-¹³C]propionate and the formation of acetate equally labeled in the C₁ and C₂ position. In the absence of sulfate, [3-¹³C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-¹³C]propionate partly into the higher fatty acid 2-methyl[2,3-¹³C]butyrate during the simultaneous degradation of [2,3-¹³C]propionate and butyrate. [4,5-¹³C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.     

Reductive Carboxylation of Propionate to Butyrate in Methanogenic Ecosystems

During the batch degradation of sodium propionate by the anaerobic sludge from an industrial digestor, we observed a significant amount of butyrate formation. Varying the initial propionate concentrations did not alter the ratio of maximal butyrate accumulation to initial propionate concentration within a large range. By measuring the decrease in the radioactivity of [1-¹⁴C]butyrate during propionate degradation, we estimated that about 20% of the propionate was converted to butyrate. Labeled butyrate was formed from [1-¹⁴C]propionate with the same specific radioactivity, suggesting a possible direct pathway from propionate to butyrate. We confirmed this hypothesis by nuclear magnetic resonance studies with [¹³C]propionate. The results showed that [1-¹³C]-, [2-¹³C]-, and [3-¹³C]propionate were converted to [2-¹³C]-, [3-¹³C]-, and [4-¹³C]butyrate, respectively, demonstrating the direct carboxylation on the carboxyl group of propionate without randomization of the other two carbons. In addition, we observed an exchange reaction between C-2 and C-3 of the propionate, indicating that acetogensis may proceed through a randomizing pathway. The physiological significance and importance of various metabolic pathways involved in propionate degradation are discussed, and an unusual pathway of butyrate synthesis is proposed.     

Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [¹³C₁]propionate and [¹³C₃]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[²H₂]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Announcement

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

Biosynthesis of (−)-ent-kaurenoic acid in Smallanthus sonchifolius and its effect against microbial biofilms

The biosynthetic pathway of (–)-ent-kaurenoic acid (1) was investigated by incorporation of 1-d-¹³C-glucose in Smallanthus sonchifolius (Asteraceae) plantlets. The ¹³C-enrichment pattern indicated that methylerythritol-4-phosphate (MEP) pathway is the biosynthetic pathway involved in diterpenoid biosynthesis. Our studies in S. sonchifolius reinforce that the biosynthesis of different classes of terpenes should not be compartmentalized into cytosol and plastids. Additionally, (–)-ent-kaurenoic acid showed antimicrobial activity against Staphylococcus aureus biofilm.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

Growth of Syntrophic Propionate-Oxidizing Bacteria with Fumarate in the Absence of Methanogenic Bacteria

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. ¹³C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO₂. Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.     

Participation of propionate in cholesterol biosynthesis by rat liver

Incorporation of (2¹⁴C) propionate into cholesterol was demonstrated using rat liver slices and homogenates. The incorporation of (2¹⁴C) propionate was greater than that of (2¹⁴C) acetate. Using the same liver homogenate preparation (2 ¹⁴C) succinate and (2¹⁴C) pyruvate were incorporated into cholesterol to a lesser extent than (2¹⁴C) acetate and (2 ¹⁴C) propionate. Addition of unlabeled acetate failed to dilute the incorporation of (2 ¹⁴C) propionate. Incorporation of the 2 and 3 carbon atoms pf propionate were equal; little incorporation of the 1 carbon atom was demonstrable. These results indicate that propionate is an excellent source of 2 carbon units for isoprenoid biosynthesis; the intermediary pathway does not involve a common acetate pool nor can these results be satisfactorily explained by citric acid intermediary metabolism.