relA Gene control of bacterial glycogen synthesis.

Starvation of Escherichia coli K12 for an amino acid results in the stimulation of bacterial glycogen synthesis in cells containing the relA+ gene, but not in cells carrying the relA- allele. Similarly, a large difference in glycogen content is demonstrable between relA+ and relA- cells in stationary phase. It is concluded that guanosine 5',3' -bis(diphosphate) (ppGPP) or some related relA -dependent metabolite is involved in the regulation of bacterial glycogen synthesis. Detection of significant basal levels of glycogen in a relA- strain of E. coli and in unstarved relA+ C. coli indicates that relA control is not absolutely required for glycogen synthesis but serves as a signal for modulation in response to nutrient availability.     

O-alkyl lipid synthesis: the mechanism of the acyl dihydroxyacetone phosphate fatty acid exchange reaction

We have previously provided evidence for a mechanism by which acyl DHAP is converted enzymatically to O-alkyl DHAP. This mechanism involves, in part, the formation of an endiol of acyl DHAP, loss of the fatty acid by splitting of the DHAP carbon-1 to oxygen bond and the gain of a long chain fatty alcohol. It has been shown that acyl DHAP can exchange its fatty acid for one in the medium, presumably by the mediation of O-alkyl DHAP synthase. In the present investigation we have shown that the fatty acid which is gained by acyl DHAP in the exchange process retains both carboxyl oxygens, as predicted by our postulated mechanism. This reaction is exceptional because the usual action of acyl hydrolases is to cleave at the oxygen to acyl bond.     

Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.     

FT-IR spectrometry analysis of plasma fatty acyl moieties selective mobilization during endurance exercise

This study was designed to describe changes in plasma fatty acyl moieties during a 2-h endurance exercise. Sixteen endurance-trained subjects cycled 2 h at 55% of maximal power output and capillary blood was sampled every 15 min. Fourier-transform infrared (FT-IR) spectrometry was used to determine correlated changes between plasma fatty acyl moieties (FAM) structural characteristics and metabolic parameters (oxygen consumption, respiratory exchange ratio, glucose, lactate, TG, glycerol, and albumin). Opposite changes were found between carbohydrate and fatty acid metabolism during the second hour of exercise, i.e., a decrease of glucose and lactate concentrations while albumin, FAM, and TG increased. For fatty acid metabolism, FAM and TG did not showed the same pattern of changes at the end of exercise, i.e., TG remained constant after 90 min while FAM continued to increase. This late FAM concentration increase was correlated to the changes in albumin concentration and the nu C=C-H/nu(as) CH3 and nu(as) CH2/nu(as) CH3 ratios. These ratios clearly showed that FAM unsaturation increased while chain length decreased. It was hypothesized that PUFA from TG adipose lipolysis ketone bodies (beta-hydroxybutyric acid) from liver may have been released in higher amounts as glycogen stores became depleted after 90 min of exercise.     

Control of triglyceride synthesis by the intracellular level of long-chain acyl coenzyme A for lipid synthesis

Candida lipolytica mutants defective in acyl coenzyme A synthetase I synthesized triglyceride to a markedly less extent than did the wild-type yeast, when grown on oleic acid. The synthesis of triglyceride was controlled by the level of long-chain acyl coenzyme A available for lipid synthesis, whereas the synthesis of phospholipids was hardly affected.     

Lipid A structures containing novel lipid moieties: synthesis and adjuvant properties

Structurally well-defined immune stimulatory molecules are important components of new generation molecular vaccines. In this paper, the design and synthesis of two lipid A analogues containing an unnatural tri-lipid acyl group are described. In a totally synthetic liposomal vaccine system, these re-designed lipid A analogues demonstrate potent immune stimulatory properties including antigen specific T-cell activation.     

Specificity of the fatty acyl moieties of diacylglycerol for the activation of calcium-activated, phospholipid-dependent protein kinase

The specificity of the fatty acyl moieties of diacylglycerol for the activation of Ca2+-activated, phospholipid-dependent protein kinase was investigated. Diacylglycerol has been previously shown to activate this enzyme by increasing the affinity for Ca2+ and phospholipid, both of which are indispensable for the enzyme activation. Diacylglycerols containing at least one unsaturated fatty acid at either position 1 or 2 are fully active in this capacity, irrespective of the chain length of the other fatty acyl moiety in the range tested, C2 to C18. Diacylglycerols containing two saturated fatty acids such as dipalmitin and distearin are far less effective. Mono- and triacylglycerols and free fatty acids are totally inactive, indicating that the diacylglycerol structure is essential.     

Interdigitated lipid bilayers of long acyl chain species of cerebroside sulfate. A fatty acid spin label study

The metastable phase behavior of semi-synthetic species of cerebroside sulfate (CBS), with hydroxy and non-hydroxy fatty acids from 16 to 26 carbons in length, was compared in Li+ and K+ using differential scanning calorimetry. The structure of the metastable and various stable phases formed in the presence of these two cations was investigated using a fatty acid spin label, 16-doxylstearate. A number of stable phases with successively higher phase transition temperatures and enthalpies occur in the presence of K+ (see the preceding paper). Li+ prevents formation of the most stable phases with the highest transition temperatures and enthalpies for all species of CBS. However, it does not prevent a transition from the metastable phase to the first stable phase of the longer chain C24 and C26 species. Furthermore, it allows C24:0h-CBS to undergo a similar transition, in contrast to a high K+ concentration, which prevents it. The spin label has anisotropic motion in the metastable gel phase formed by all species of CBS on cooling from the liquid crystalline phase. The spectra resemble those in gel phase phospholipids. The spin label is partially insoluble in the most stable phases formed by all the lipids, including the unsaturated C24:1 species, preventing further elucidation of their structure using this technique. However, the spin label is soluble in the first stable phase formed on cooling by the longer chain C24:0 and C26:0-CBS in Li+ and K+ and by C24:0h-CBS in Li+, and is motionally restricted in this phase. The motional restriction is similar to that observed in the mixed interdigitated bilayers of asymmetric species of phosphatidylcholine and fully interdigitated bilayers formed by symmetric phospholipids. It strongly suggests that the highly asymmetric long chain species of CBS form a mixed interdigitated bilayer in their first stable gel phases while the metastable phase of these and the shorter chain lipids may be partially interdigitated. The metastable phase of C24:1-CBS is more disordered suggesting that it may not be interdigitated at all. Thus the results suggest that (i) the hydroxy fatty acid inhibits but does not prevent formation of a mixed interdigitated bilayer by long chain species of CBS, (ii) an increase in non-hydroxy fatty acid chain length from 24 to 26 carbons promotes it, and (iii) a cis double bond probably prevents any form of interdigitation. These results may be relevant to the physiological and pathological roles of these structural modifications of CBS.     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of poiymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2. trans-4. trans-6. cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C1 8:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viviae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the 18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the poly-unsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.     

Independence of cyclic AMP and relA gene stimulation of glycogen synthesis in intact Escherichia coli cells.

Previous studies from our laboratory established that in Escherichia coli, glycogen synthesis is regulated by both the relA gene, which mediates the stringent response, and by cyclic AMP. However, those studies raised the question of whether this dual regulatory system functions in an independent or a dependent manner. We show here that this regulation is independent, i.e., each regulatory process can express its action in the absence of the other. Triggering the stringent response by amino acid starvation increased glycogen synthesis even in mutants lacking the ability to synthesize cyclic AMP or lacking cyclic AMP receptor protein; and cyclic AMP addition stimulated glycogen synthesis in relA mutant strains. We also show that physiological concentrations of GTP inhibit ADP-glucose synthetase (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), the rate-limiting enzyme of bacterial glycogen synthesis, in vitro. Because the stringent response is known to cause an abrupt decrease in the cellular level of GTP, modulation of ADP-glucose synthetase activity by this nucleotide could account for a substantial portion of the step-up in the cellular rate of glycogen synthesis observed when the stringent response is triggered.