Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli.

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli.     

Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The GlpT system for sn-glycerol-3-phosphate transport in Escherichia coli is shown to catalyze a rapid efflux of Pi from the internal phosphate pools in response to externally added Pi or glycerol-3-phosphate. A glpR mutation, which results in constitutive expression of the GlpT system, is responsible for this rapid Pi efflux and the arsenate sensitivity of several laboratory strains, including the popular strain C600. Glucose and other phosphotransferase system sugars inhibit Pi efflux by repressing glpT expression.     

Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli

Summary Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map.Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.     

Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.

Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell. Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria. This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges. The long-pitch helices represented chains of acyltransferase dimers. Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules. Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane. The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm. Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains. Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.     

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.     

Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli.

Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.     

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

Summary The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.     

Phospholipid dependence of homogeneous, reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli

A novel mixed micelle assay for the sn-glycerol-3-phosphate acyltransferase of Escherichia coli was developed using the nonionic detergent octaethylenegly-coldodecyl ether. The assay permitted investigation of the phospholipid dependence of enzyme activity at phospholipid/detergent ratios of 5:1 (w/w) to 2:1 depending on the phospholipid employed. The higher ratio yielded maximal activity when E. coli phospholipids were used; the lower ratio was observed with cardiolipin(E. coli). Phosphatidylglycerol(E. coli) and phosphatidylethanolamine(E. coli) also restored enzyme activity. Activation by phosphatidylethanolamine(E. coli) was pH-dependent and relatively inefficient. The synthetic, disaturated (1,2-palmitoyl)phosphatidylglycerol reconstituted only 25% of the total enzyme activity as that observed with the monounsaturated (1-palmitoyl, 2-oleoyl) species. Full activation of enzyme was achieved with (1,2-dioleoyl)phosphatidylglycerol. Phosphatidylcholine and phosphatidic acid were unable to reconstitute enzyme activity. Chromatographic sizing of the sn-glycerol-3-phosphate acyltransferase, following reconstitution in cardiolipin(E. coli)/octaethyleneglycoldodecyl ether mixed micelles, suggested that the monomeric form of the enzyme was active.     

The triose-phosphate site of homogeneous reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The sn-glycerol-3-phosphate (glycerol-phosphate) acyltransferase of Escherichia coli was purified to near homogeneity and its activity reconstituted with phospholipids (Green, P.R., Merrill, A.M., Jr. and Bell, R.M. (1981) J. Biol. Chem. 256, 11151-11159). The competency of glycerol-P analogues to serve as inhibitors and as substrates was investigated. Dihydroxyacetone-P, ethyleneglycol-P, 1,3-propanediol-P, 3,4-dihydroxybutylphosphonate and DL-glyceraldehyde-3-P were inhibitors of the reconstituted purified glycerol-phosphate acyltransferase. The kinetics of inhibition, while formally of the mixed type, most closely resembled that of a simple competitive inhibition with respect to glycerol-3-P. Inorganic phosphate was also found to be a competitive inhibitor. All of the glycerol-3-P analogues except DL-glyceraldehyde-3-P were substrates. Of these, dihydroxyacetone-P proved to be the best substrate. The secondary hydroxyl was not necessary for activity. Glycerol-phosphate acyltransferase catalyzed the hydrolysis of palmitoyl-CoA in the presence of DL-, but not D-glyceraldehyde-3-P. This suggests that the gem diol of L-glyceraldehyde-3-P may be a substrate, and that the acylated adduct may be unstable. The enzyme was inactivated by phenylglyoxal and butanedione, suggesting that arginine may be at or near the active site.     

Role of spermidine in the activity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli

The integral membrane protein, sn-glycerol-3-phosphate acyltransferase, catalyzes the first committed step in phospholipid synthesis, and both acyl-CoA and acyl-acyl carrier protein can be used as acyl donors in this reaction. We found that spermidine increased the specific activity of the acyltransferase when either substrate was used as the acyl donor. Magnesium, as well as other cations, also increased acyltransferase activity but were not nearly as effective as spermidine. Two roles for spermidine in this reaction were deduced from our data. First, spermidine dramatically lowered the K_m for glycerol 3-phosphate resulting in an overall rate enhancement when either substrate was used as the acyl donor. This effect was attributed to the modification of the acyl-transferase environment due to the binding of spermidine to membrane phospholipids. A second effect of spermidine was evident only when acyl-acyl carrier protein was used as substrate. Using this acyl donor, a pH optimum of 7.5 was found in the absence of spermidine, but in its presence, the pH optimum was shifted to 8.5. Between pH 7.5 and 8.5, palmitoyl-acyl carrier protein undergoes a conformational change to a more expanded, denatured state and its activity in the acyltransferase assay decreases dramatically. Spermidine restored the native conformation of palmitoyl-acyl carrier protein at pH 8.5, thus accounting for the majority of rate enhancement observed at elevated pH.     

Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli.

It is known that in Escherichia coli two dehydrogenases of the flavoprotein kind can participate in the transfer of hydrogens from sn-glycerol 3-phosphate (G3P) to nitrate and that possession of either enzyme is sufficient to permit anaerobic growth on glycerol as carbon source and nitrate as hydrogen acceptor. Results from this study show that under such a growth condition a protein with light-absorption characteristics of cytochrome b1 is induced. If G3P, nitrate, and adenosine diphosphate are added anaerobically to a particulate fraction prepared from these cells, four reactions can be detected: (i) the reduction of the cytochrome b1-like protein, (ii) the formation of dihydroxyacetone phosphate (DHAP), (iii) the formation of nitrite, and (iv) the generation of adenosine 5'-triphosphate (ATP). The anaerobic G3P dehydrogenase system can yield an ATP-DHAP (or ATP-nitrite) molar ratio of about 0.2, whereas the aerobic G3P dehydrogenase system can yield a corresponding ratio of about 0.3. The hydrogen transfer activity is sensitive to respiratory inhibitors such as cyanide, Rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Regulation of ugp, the sn-glycerol-3-phosphate transport system of Escherichia coli K-12 that is part of the pho regulon.

The expression of the ugp-dependent sn-glycerol-3-phosphate transport system that is part of the pho regulon was studied in mutants of Escherichia coli K-12 containing regulatory mutations of the pho regulon. The phoR and phoST gene products exerted a negative control on the expression of ugp. Induction of the system was positively controlled by the phoB, phoM, and phoR gene products. Using a ugp-lacZ operon fusion, we showed that the ugp and phoA genes were coordinately derepressed and repressed.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed.