Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant

Escherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coli membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations.     

DHA system mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418.

In Klebsiella pneumoniae NCIB 418, the pathways normally responsible for aerobic growth on glycerol and sn-glycerol 3-phosphate (the glp system) are superrepressed. However, aerobic growth on glycerol can take place by the intervention of the NAD-linked glycerol dehydrogenase and the ATP-dependent dihydroxyacetone kinase of the dha system normally inducible only anaerobically by glycerol or dihydroxyacetone. Conclusive evidence that the dha system is responsible for both aerobic and anaerobic dissimilation of glycerol was provided by a Tn5 insertion mutant lacking dihydroxyacetone kinase. An enzymatically coupled assay specific for this enzyme was devised. Spontaneous reactivation of the glp system was achieved by selection for aerobic growth on sn-glycerol 3-phosphate or on limiting glycerol as the sole carbon and energy source. However, the expression of this system became constitutive. Aerobic operation of the glp system highly represses synthesis of the dha system enzymes by catabolite repression.     

Dehydrogenation of a phosphonate substrate analogue by glycerol 3-phosphate dehydrogenase

S-(+)-3,4-Dihydroxybutylphosphonic acid, an isosteric analogue of sn-glycerol 3-phosphate, was synthesized stereospecifically and shown to be an effective substrate for rabbit muscle glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate-NAD(+) oxidoreductase, EC 1.1.1.8). Non-isosteric phosphonate analogues of sn-glycerol 3-phosphate showed neither substrate nor inhibitory activity with the enzyme.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism.

sn-Glycerol 3-phosphorothioate was found to be bacteriocidal to strains of Escherichia coli which have a functional sn-glycerol 3-phosphate transport system. This effect was manifest in strains 7 and 8, which are constitutive mutants for the utilization and transport of sn-glycerol 3-phosphate (glpRc2). Strain E15, which is considered to be wild type for the glycerol phosphate functional units, was affected by the phosphorothioate analog only under conditions that are known to induce the transport system for sn-glycerol 3-phosphate. In addition, another strain of E. coli, strain 6, which is isogenic with strain E15 but has an impaired sn-glycerol 3-phosphate transport system (glpT13), was not affected by similar concentrations of sn-glycerol 3-phosphorothioate. Transport studies in which [3H]glycerol phosphate and its phosphorothioate analog were used demonstrated that the latter compound was taken up via the specific active transport system for sn-glycerol 3-phosphate; the Km values were 9 and 11 microM, respectively. The rates of macromolecular synthesis were found to be inhibited severely by sn-glycerol 3-phosphorothioate at a concentration at which sn-glycerol 3-phosphate had no effect (5 microM). At a lower concentration of the analog (0.5 microM), the rates of protein synthesis and RNA synthesis (52 and 58% below control values after 90 min, respectively) were more sensitive than the rates of DNA synthesis and cell wall synthesis (18% below control values after 3 h for DNA; transient decrease in the cell wall values after 90 min). The levels of the nucleoside triphosphates were not affected by the presence of the phospholipid precursor or its analog at a concentration of 5 microM. The phospholipid composition was significantly altered in the presence of bacteriocidal concentrations (5 microM) of sn-glycerol 3-phosphorothioate. The amount of phosphatidylglycerol in the membranes decreased from 13.5 to 3.5%. Concomitant with this decrease in phosphatidylglycerol content was a fourfold increase in the 32P content of cardiolipin (from 6.8 to 24.2%), whereas the phosphatidylethanolamine content showed only a minor reduction (8%) after 3 h. The rates of synthesis of all of the phospholipids decreased in the presence of 5 microM sn-glycerol 3-phosphorothioate, with the most significant effects observed for phosphatidylglycerol (63% after 3 h). Phosphatidylglycerol showed increased rates of turnover after 90 min (21%) and 3 h (11%), with concomitant increases in the levels of cardiolipin of more than twofold. Our data suggest that a considerably greater proportion of phosphatidylglycerol turnover may be recover in cardiolipin than is metabolized via other pathways (e.g., the membrane-derived oligosaccharide pathway).     

Caulobacter cresentus mutant defective in membrane phospholipid synthesis.

To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.     

Heterogeneity of the sn-glycerol 3-phosphate pool in isolated hepatocytes, demonstrated by the use of deuterated glycerols and ethanol.

Hepatocytes were isolated from female rats and incubated with [1,1,3,3-2H4]glycerol or [2-2H]glycerol. The deuterium excess in phosphatidylcholines, sn-glycerol 3-phosphate and other organic acids was determined by g.l.c./mass spectrometry. The unlabelled fraction of the major phosphatidylcholines decreased exponentially, and the turnover was not changed by the presence of ethanol. The relative contribution of the two deuterated glycerols was about the same in the major phosphatidylcholine as in sn-glycerol 3-phosphate, indicating that formation by acylation of dihydroxyacetone phosphate is insignificant. [1,1,3,3-2H4]Glycerol had lost deuterium to a larger extent when it was incorporated in the phosphatidylcholine than when it was incorporated in sn-glycerol-3-phosphate, indicating that the phosphatidylcholines are formed from a separate pool of sn-glycerol 3-phosphate. Deuterium at C-2 was transferred between sn-glycerol 3-phosphate molecules to about 25%. Ethanol decreased the extent of deuterium transfer, the extent of glycerol uptake and the loss of deuterium at C-1 and C-3 in sn-glycerol 3-phosphate. The results indicate that the oxidation to dihydroxyacetone phosphate was inhibited by the NADH formed during ethanol oxidation. [2-2H]Glycerol also labelled an alcohol dehydrogenase substrate, malate and lactate, indicating oxidation of sn-glycerol 3-phosphate in the cytosol. The two acids appeared to be formed in reductions with different pools of NADH.     

Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon

The promoter-proximal gene (glpT) of the glpT-glpQ operon of Escherichia coli encodes a membrane permease responsible for active transport of sn-glycerol 3-phosphate. Promoter-distal glpQ encodes a periplasmic protein which is not required for active transport of sn-glycerol 3-phosphate (Larson, T.J., Schumacher, G., and Boos, W. (1982) J. Bacteriol. 152, 1008-1021). This periplasmic protein has now been identified as a phosphodiesterase which hydrolyzes glycerophosphodiesters into sn-glycerol 3-phosphate plus alcohol. The enzyme exhibited broad substrate specificity with respect to the alcohol moiety; sn-glycerol 3-phosphate was released from glycerophosphoethanolamine, glycerophosphocholine, glycerophosphoglycerol, and bis(glycerophospho)glycerol. The enzyme was specific for glycerophosphodiesters; bis(p-nitrophenyl)phosphate, a substrate for other phosphodiesterases, was not hydrolyzed. In a coupled spectrophotometric assay utilizing sn-glycerol 3-phosphate dehydrogenase and NAD, apparent activity was optimal at pH 9 and was stimulated by Ca2+. The substrates of the phosphodiesterase had no affinity for the glpT-encoded active transport system. Thus, the glpQ gene product expands the catabolic capability of the glp regulon to include a variety of glycerophosphodiesters.     

Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): pathway and properties of enzymes of sn-glycerol 3-phosphate formation

The enzymatic pathway for the synthesis of sn-glycerol 3-phosphate was investigated in developing groundnut seeds (Arachis hypogaea). Glycerol-3-phosphate dehydrogenase was not detected in this tissue but an active glycerokinase was demonstrated in the cytosolic fraction. It showed an optimum pH at 8.6 and positive cooperative interactions with both glycerol and ATP. Triosephosphate isomerase and glyceraldehyde-3-phosphate phosphatase were observed mainly in the cytosolic fraction while an active glyceraldehyde reductase was found mainly in the mitochondrial and microsomal fractions. The glyceraldehyde 3-phosphate phosphatase showed specificity and positive cooperativity with respect to glyceraldehyde 3-phosphate. The glyceraldehyde reductase was active toward glucose and fructose but not toward formaldehyde and showed absolute specificity toward NADPH. It is concluded that in the developing groundnut seed, sn-glycerol 3-phosphate is synthesized essentially by the pathway dihydroxyacetone phosphate----glyceraldehyde 3-phosphate Pi----glyceraldehyde NADPH----glycerol ATP----glycerol 3-phosphate. All the enzymes of this pathway showed activity profiles commensurate with their participation in triacylglycerol synthesis which is maximal during the period 15-35 days after fertilization. Glycerokinase appears to be the rate-limiting enzyme in this pathway.     

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.     

Phenethyl alcohol inhibition of sn-glycerol 3-phosphate acylation in Escherichia coli.

In vivo and in vitro experiments were performed to determine how phenethyl alcohol (PEA) inhibits phospholipid synthesis in Escherichia coli. This drug drastically reduced the rate of incorporation of sn-glycerol 3-phosphate into the phospholipids of an sn-glycerol 3-phosphate auxotroph. PEA also reduced the rate of fatty acid incorporation into the phospholipids of a fatty acid auxotroph. The kinetics of PEA inhibition of the rate of incorporation of sn-glycerol 3-phosphate were almost identical to those of PEA inhibition of the rate of fatty acid incorporation into phospholipids. The in vivo experiments suggested that the rate-limiting step(s) in phospholipid biosynthesis inhibited by PEA is at the level of the acylation of sn-glycerol 3-phosphate or beyond this step. PEA inhibited the sn-glycerol 3-phosphate acyltransferase with either palmitoyl coenzyme A or palmitoyl-acyl carrier protein as the acyl donor. This drug, however, had no effect on the cytidine 5'-diphosphate-diglyceride:glycerol 3-phosphate phosphatidyl transferase, cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase, and acyl coenzyme A:lysophatidic acid acyltransferase. The in vitro findings suggested that PEA inhibits phospholipid synthesis primarily at the level of sn-glycerol 3-phosphate acyltransferase.     

Metabolism of D-fructose in Aerobacter aerogenes: analysis of mutants lacking D-fructose 6-phosphate kinase and D-fructose 1,6-diphosphatase

The relative significance of the pathways for the conversion of d-fructose to d-fructose 1,6-diphosphate via d-fructose 1-phosphate or d-fructose 6-phosphate in Aerobacter aerogenes PRL-R3 was assessed by observing growth patterns of mutants lacking either d-fructose 6-phosphate kinase or d-fructose 1,6-diphosphatase. The mutant lacking d-fructose 6-phosphate kinase grew well on d-fructose or glycerol but not on d-glucose, whereas the mutant lacking d-fructose 1,6-diphosphatase grew on d-glucose but not on d-fructose or glycerol. The data indicate that the pathway of d-fructose metabolism is primarily through d-fructose 1-phosphate rather than d-fructose 6-phosphate.     

Mechanisms for the effects of ethanol on hepatic phosphatidate phosphohydrolase.

1. The effects of the intramuscular administration of glycerol and dihydroxyacetone (40mmol per kg body wt.), sorbitol and glucose (20mmol per kg body wt.) or NaCl (1.5mmol per kg body wt. in 10ml of water per kg body wt.) were investigated on soluble phosphatidate phosphohydrolase and certain metabolites in rat liver. 2. The effects of ethanol and glycerol on phosphatidate phosphohydrolase were also studied in isolated perfused livers. 3. The administration of glycerol, sorbitol and dihydroxyacetone in vivo increased hepatic phosphatidate phosphohydrolase activity by 137, 63 and 32% respectively in 4h. 4. A significant positive correlation was found between the hepatic sn-glycerol 3-phosphate concentration and phosphatidate phosphohydrolase after the administration of various substrates in vivo. 5. The soluble phosphatidate phosphohydrolase activity tended to increase during perfusions of isolated rat livers without added substrates, and neither ethanol nor glycerol produced additional effects. 6. The activity of soluble phosphatidate phosphohydrolase was 2.5 times higher in the livers of hyperthyroid rats than in normal rats. This activity was not influenced by intragastric ethanol or glycerol administration, nor was the concentration of sn-glycerol 3-phosphate changed by these compounds. 7. It is concluded that the ethanol-induced increase in hepatic phosphatidate phosphohydrolase may at least in part be mediated by the hepatic concentration of metabolites, probably by the concentration of sn-glycerol 3-phosphate.     

The interconversion of diacylglycerol and phosphatidylcholine during triacylglycerol production in microsomal preparations of developing cotyledons of safflower (Carthamus tinctorius L.).

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalyse the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. Under these conditions the radioactive glycerol in sn-glycerol 3-phosphate accumulates in phosphatidic acid, phosphatidylcholine, diacyl- and tri-acylglycerol. The incorporation of glycerol into phosphatidylcholine is via diacylglycerol and probably involves a cholinephosphotransferase. The results show that the glycerol moiety and the acyl components in phosphatidylcholine exchange with the diacylglycerol during the biosynthesis of diacylglycerol from phosphatidic acid. The continuous reversible transfer of diacylglycerol with phosphatidylcholine, which operates during active triacylglycerol synthesis, will control in part the polyunsaturated-fatty-acid quality of the final seed oil.     

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.     

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.     

Transport of 3,4-dihydroxybutyl-1-phosphonate, an analogue of sn-glycerol 3-phosphate.

3,4-Dihydroxybutyl-1-phosphonate (DHBP), an analogue of glycerol 3-phosphate, is actively transported by the sn-glycerol 3-phosphate transport system of Escherichia coli strain 8. The Km for the transport of DHBP is 200 microM.     

Microcalorimetric monitoring of growth of Saccharomyces cerevisiae: osmotolerance in relation to physiological state.

The importance of the physiological state of a culture of Saccharomyces cerevisiae for tolerance to sudden osmotic dehydration was studied, and it was investigated whether specific osmotolerance factors were demonstrable. The microcalorimeter was used to monitor growth, and different physiological states of the culture were selected and their osmotolerance was tested. In addition to cells in the stationary phase, cells from the transition phase between respirofermentative and respiratory catabolism were osmotolerant. S. cerevisiae exhibited ever-changing metabolism during batch growth on either glucose or ethanol as the carbon source. Instantaneous heat production per biomass formation (dQ/dX) and specific activity of sn-glycerol 3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) were shown to differ for different physiological states. Neither high respiratory activity nor low total cellular activity, nor factors involved in osmoregulation, i.e., intracellular glycerol or activity of GPDH, correlated with the osmotolerant phenotype.     

The formation of phosphatidic acid de novo: a comparison of activities in neuronal nuclei and microsomes isolated from immature rabbit cerebral cortex

The formation of phosphatidic acid from sn-glycerol 3-phosphate was studied in neuronal nuclear fraction N1 and a microsomal fraction P3, isolated from cerebral cortices of 15-day-old rabbits. Two assays were used, employing dithiothreitol, MgCl2, NaF and (A) sn-glycerol 3-phosphate, [14C]oleate, ATP and CoA or (B) sn-[3H]glycerol 3-phosphate and oleoyl-CoA. In both assays fraction N1 had specific rates of phosphatidic acid labelling (expressed per mumol phospholipid in the fraction) which were 5- to 6-times the corresponding values for P3. In contrast to N1, the formation of phosphatidic acid by fraction P3 was more sensitive to inhibition at high concentrations of oleoyl-CoA and was greatly dependent upon the presence of NaF. In the absence of this salt, P3 showed decreased phosphatidate formation and increased levels of radioactive monoacylglycerols. Using cerebral cortex, rough (R) and smooth (S) microsomal fractions were prepared, as was a microsomal fraction P from isolated nerve cell bodies. P had specific rates of phosphatidic acid labelling which were 2-3 times the values for P3, but were about 50% of the N1 values. This indicates a concentration of phosphatidate synthesis in the nucleus within the nerve cell. Specific rates for fraction R were higher and were similar to those of N1. In S, P3 and R the specific rates of phosphatidic acid synthesis paralleled specific RNA contents and indicated a location for phosphatidic acid synthesis within the rough endoplasmic reticulum.     

The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.