Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates.

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [(14)C]mevalonate (MVA), [(14)C]IPP, and [(14)C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [(14)C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [(14)C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action.     

Studies of the in vivo metabolism of mevalonic acid in the neonatal chick

After 4 hr of the intraperitoneal injection of different doses of (R)-[5-14C]mevalonic acid (MVA), its incorporation into nonsaponifiable and saponifiable lipids was maximal in neonatal chick kidneys and liver, and minimal in brain, spinal cord and skin. Using 14CO2 production from [5-14C]MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that about 11% of MVA was in vivo metabolized by this pathway in nonmammalian species. Kidneys presented the maximal ability to incorporate MVA into nonsaponifiable and saponifiable lipids at any time considered (15-750 min). The percentage of radioactivity recovered as saponifiable lipids in liver and kidney decreased after 12 hr the injection of MVA. Although the absolute amounts of 14C incorporated in both derivatives were much less in brain, spinal cord and skin than in liver and kidneys, the relative percentages found in the saponifiable fraction were clearly higher in the former tissues, especially in the spinal cord.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

Biosynthesis of avenacins and phytosterols in roots of Avena sativa cv. Image

In keeping with the proposal that avenacin biosynthesis is restricted to the tips of primary roots of oat seedlings, the incorporation of radioactivity from R-[2-(14)C]mevalonic acid (MVA) into avenacins and beta-amyrin by serial sections of primary roots was found to be more-or-less restricted to root tip sections. Squalene synthase (SQS) (EC 2.5.1.21) and 2,3-oxidosqualene:beta-amyrin cyclase (OS beta AC) (EC 5.4.99) were also most active in these sections. The incorporation of radiolabel from R-[2-(14)C]MVA into cycloartenol and 24-methylene cycloartanol by, and the 2,3-oxidosqualene:cycloartenol cyclase (OSCC) (EC 5.4.99) activity in, the various serial sections were consistent with phytosterol biosynthesis occurring in all the sections of the root with some tailing-off in the rate of synthesis in the more distal sections.     

Formation of terpenoid products in Ginkgo biloba L. cultivated cells

Ginkgolides are diterpenes arising from the terpenoid precursor: geranylgeranyl pyrophosphate (GGPP). Incorporation of [1-¹⁴C] isopentenylpyrophosphate ([1-¹⁴C]IPP) into GGPP was monitored throughout the cultivation cycle of G. biloba L. cultivated cells. Because incorporation of [1-¹⁴C]IPP into GGPP had never been monitored in G. biloba, in either the whole plant or cultivated cell system, modifications to existing protocols were necessary. Modifications consisted of extracting the cells with an extraction buffer supplemented with Triton-X-100. Farnesylpyrophosphate (FPP) was the major product formed. The amount of GGPP detected was about one tenth that of FPP.Abbreviations CHAPS 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane-sulphonate - DTT [1-4 dithiothreitol] - FPP farnesylpyrophosphate - GGPP geranylgeranylpyrophosphate - IPP [1-¹⁴C] isopentenylpyrophosphate - PVPP polyvinylpolypyrrolidone - Tris Tris(hydroxymethyl)aminomethane     

Effect of gibberellic acid on sterol production in Corylus avellana seeds

Gibberellic acid-induced germination of hazel seeds was accompanied by little change in the sterol content of the cotyledons. Dormant and germinating cotyledons rapidly incorporated [2-¹⁴C]MVA into squalene which was slowly converted to sterols. Gibberellin treatment induced an increase in the incorporation of [2-¹⁴C]MVA into cotyledon esterified sterols. An increase in free sterols occurred in the germinating embryonic axes, with increased relative amounts of stigmasterol and campesterol in the free 4-desmethylsterols. Germination was accompanied by increased incorporation of [2-¹⁴C]MVA into free and esterified sterols in the embryonic axes.     

The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.     

Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzyme stereospecificity

A prenyltransferase purified from the commercial rubber tree, Hevea brasiliensis, that elongates existing cis-polyisoprene rubber molecules also catalyzes the formation of all trans-farnesyl pyrophosphate (t,t-FPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In assays of the latter activity trans-geranyl pyrophosphate is the only other product identified. In contrast to this limited addition of IPP to DMAPP, we measured 7000 additions of isoprene per rubber molecule in a previous titration of active allylic ends of rubber molecules by purified prenyltransferase (Light, D. R., and Dennis, M. S. (1989) J. Biol. Chem. 264, 18589-18597). In order to confirm that purified prenyltransferase extensively elongates rubber molecules, doubly labeled [1-14C]isopentenyl [U-32P]pyrophosphate ([14C,32P]IPP) was synthesized. Using this reagent we show that both prenyltransferase purified from H. brasiliensis and prenyltransferase purified from avian liver (FPP synthase) add greater than 15 isoprene units to existing rubber molecules, consistent with the previous titration data. For confirmation that the prenyltransferase purified from H. brasiliensis adds isoprene units to rubber to make cis-polyisoprene, chirally tritiated [14C]IPP ([14C,2S-3H]IPP) was synthesized. Retention of the tritium label in FPP synthesized from [14C,2S-3H]IPP and DMAPP, geranyl pyrophosphate, or neryl pyrophosphate by prenyltransferase from H. brasiliensis or avian liver confirms trans addition to these substrates. In contrast, when [14C,2S-3H]IPP is incubated with serum-free rubber particles and prenyltransferase purified from H. brasiliensis, avian liver, or yeast, no tritium is incorporated into the rubber particles indicating cis addition. Thus, rubber particles have the ability to alter the stereoselective removal of the 2R-prochiral proton in favor of the removal of the 2S-prochiral proton. This apparent inversion of carbon 2 of IPP during the proton abstraction step by rubber particles represents a novel example of a switch in enzyme stereospecificity. In addition to being enzymatically similar to other prenyltransferases, rubber transferase also appears to be related immunologically to FPP synthases, since polyclonal antibodies to the H. brasiliensis prenyltransferase cross-react with the purified yeast prenyltransferase. In order to investigate potential primers of greater molecular weight than that of FPP, cis-undecaprenyl pyrophosphate (C55PP) was synthesized. C55PP stimulates the incorporation of [14C]IPP into rubber particles suggesting that it may prime new rubber molecules. However, in contrast to DMAPP, C55PP is not incorporated into any detectable products when incubated with prenyltransferase and [14C]IPP in the absence of rubber particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereochemistry of phytoene biosynthesis by isolated chloroplasts

The incorporation of [2-(14)C,(5R)-5-(3)H(1)]MVA* and [2-(14)C,5-(3)H(2)]MVA into geranylgeraniol and phytoene by a preparation of ;non-aqueous' bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.     

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-¹⁴C] and MVA-[2-¹⁴C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[¹⁴C] and terpinen-4-ol-[¹⁴C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-¹⁴C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.     

Incorporation of [14C]carbon dioxide and [2-14C]mevalonic acid into terpenoids of higher plants during chloroplast development

1. The incorporation of (14)CO(2) and dl-[2-(14)C]mevalonic acid into various terpenoids in developing chloroplasts in a number of seedlings has been studied. 2. beta-Carotene and phytol (from chlorophyll) tend to be heavily labelled from (14)CO(2), whereas sterols and beta-amyrin are only slightly labelled; with dl-[2-(14)C]mevalonic acid the situation is reversed. 3. The incorporation of (14)CO(2) into terpenoids is dependent on the stage of chloroplast development, whereas that of mevalonic acid is independent of chloroplast development. 4. The uptake of (14)CO(2) into beta-carotene and phytol in mature chloroplasts is very low in monocotyledons but somewhat greater in dicotyledons. 5. The results are discussed in relation to the view that terpenoid biosynthesis in developing chloroplasts is regulated by a combination of enzyme segregation and specific membrane permeability.     

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.     

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

Rubber production in guayule: determination of rubber producing potential

Optimum conditions for the rapid, efficient, nondestructive determination of rubber producing potential in guayule (Parthenium argentatum) were established. The rubber producing potential may be defined as the ability of the plant material to synthesize rubber from a precursor under specified conditions. To achieve this, stem slices taken from the first 5 centimeters of branches were incubated with [¹⁴C]acetate as precursor in 0.1 molar phosphate buffer (pH 6.5) at 26°C for 16 hours in the light. The ¹⁴C from labeled acetate and acetyl coenzymeA were efficiently incorporated into rubber whereas the ¹⁴C from both mevalonic acid (MVA) and isopentenylpyrophosphate (IPP) were poorly incorporated. Incorporation of 68.6% of the ¹⁴C from labeled IPP into the acetone extractable material suggests that most of the IPP was channeled down the lower terpenoid branch of the polyisoprene biosynthetic pathway. The incorporation of ¹⁴C from labeled acetate into rubber was most efficient at temperatures between 20 and 25°C. The rubber producing potential was also found to be dependent on light intensity. The roots which represent about one-third of the plant biomass not only had the highest rubber producing potential but also contained the highest amount of rubber (7.6%), indicating that the root system could be a major source of rubber. The mature stem bark also had a high rubber content and rubber producing potential, whereas the young stem had a low rubber content and a lower potential for producing rubber. The leaves showed little potential to incorporate labeled acetate into rubber and no more than 0.5% rubber was found in guayule leaves.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Metabolic engineering for the microbial production of carotenoids and related products with a focus on the rare C50 carotenoids

Carotenoids, a subfamily of terpenoids, are yellow- to red-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived from isopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.     

Metabolism of triacylglycerol in Mycobacterium smegmatis.

Mycobacterium smegmatis cells incorporated [1-14C]oleic acid into triacylglycerols (TG) from the medium more rapidly than shorter chain fatty acids, caprilic and butyric acids. This incorporation was inhibited more strongly by 10(-3) M N-ethylmaleimide than by 10(-3) M KCN. [14C]TG in the bacterial cells was utilized when the cells were in poor nutritional conditions, such as phosphate buffer (pH 7.0) containing oleic acid. Accumulation of TG was observed in the cells at late stages of growth. Diglyceride acyltransferase [EC 2.3.1.20] activity was detected in a cell-free extract from this bacterium. The pH optimum of this enzyme was between pH 7 and 9. F- and Tween 20 showed remarkable enhancing and inhibitory effects, respectively.