Co-Regulation in Escherichia coli of a Novel Transport System for sn-Glycerol-3-Phosphate and Outer Membrane Protein Ic (e, E) with Alkaline Phosphatase and Phosphate-Binding Protein

Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for sn-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the sn-glycerol-3-phosphate transport system (ugp⁺ mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different ugp⁺ mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151–157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the sn-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new sn-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.     

Characterization of the ugp region containing the genes for the phoB dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary The ugp structural genes, coding for the pho regulon dependent sn-glycerol-3-phosphate transport system, were cloned in pBR322 and characterized. The expression of the cloned ugp system was phoB dependent. Cells containing the ugp plasmid overproduced the G3P binding protein upon phosphate starvation. Tn5 mutagenesis of the cloned DNA revealed that the ugp genes are organized in two separate operons which comprise at least four genes: ugpB and ugpD constitute one operon, ugpA and ugpC constitute the other. The structural gene for the G3P binding protein (G3PBP) is ugpB.The ugpC gene product was also synthesized in minicells as a polypeptide, with an apparent molecular weight of 40,000. No gene products could be assigned to the ugpA and ugpD genes. Hybridization experiments allowed the physical characterization of 20 kb of DNA adjacent to the ugp genes on the E. coli chromosome including the liv genes.     

Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.     

Escherichia coli cytosolic glycerophosphodiester phosphodiesterase (UgpQ) requires Mg2+, Co2+, or Mn2+ for its enzyme activity

Escherichia coli cytosolic glycerophosphodiester phosphodiesterase, UgpQ, functions in the absence of other proteins encoded by the ugp operon and requires Mg²⁺, Mn²⁺, or Co²⁺, in contrast to Ca²⁺-dependent periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophosphodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ.     

Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli

Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the K_m of the transport system without influencing the V_max of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

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.     

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U-¹⁴C]-sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.     

Characterization of a binding protein involved in the transport of O-phosphorylethanolamine inPseudomonas aeruginosa

Pseudomonas aeruginosa, grown in a medium containing O-phosphorylethanolamine as the sole source of phosphate and carbon, is able to take up this ester phosphate via two kinetically distinct systems with apparent Km values of 2.5 and 55 M, respectively. An inducible O-phosphorylethanolamine-binding protein was purified to homogeneity from the shock fluid of the bacteria. It has an average molecular weight of 38 kda and an isoelectric point of 5.35. One mole of substrate was bound per mole of protein with a dissociation constant of 1.2 M. This binding protein appears very specific since neither phosphate, phosphoserine, phosphocholine,sn-glycerol-3-phosphate, 2-aminoethylphosphonic acid, nor ethanolamine could inhibit the binding of O-phosphoryl-[2-¹⁴C]ethanolamine when measured by equilibrium dialysis.     

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.     

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     

Novel stereoconfiguration in lyso-bis-phosphatidic acid of cultured BHK-cells

Lyso-bis-phosphatidic acid purified from cultured hamster kidney fibroblast cells (BHK-cells) was subjected to strong alkaline hydrolysis. The hydrolysate contained phosphorus, free glycerol, total glycerol, α-glycerophosphate, β-glycerophosphate and sn-glycerol-3-phosphate in mole ratios of 1.0:1.0:1.9:0.4:0.6:0.02. The absence of sn-glycerol-3-phosphate indicates that the backbone of this lipid has the uncommon structure of 1-sn-glycerophosphoryl-1′-sn-glycerol. Consequently, the biosynthesis and the degradation of this lipid must differ from the other known mammalian glycerophospholipids.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Purification and some properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae

An NAD-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD⁺ oxidoreductase, EC 1.1.1.8) has been isolated and purified from Saccharomyces cerevisiae by affinity and exclusion chromatography. The enzyme was purified 5100-fold to a specific activity of 158. It has a molecular weight of approximately 31,000, a pH optimum between 6.8 and 7.2, and is sensitive to high-ionic-strength salt solutions. The enzyme is most strongly inhibited by phosphate and chloride ions.     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Two new phospholipids,hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine,from the Archaea Methanosarcina barkeri

The structures of two new ether phospholipids of the methanogenic Archaea, Methanosarcina barkeri, were determined as hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine by means of chemical, chromatographic and enzymatic analyses, and fast atom bombardment-mass spectrometry. These lipids are hydroxy diether analogs of phosphatidylglycerol and phosphatidylethanolamine, respectively, with β-hydroxyarchaeol (2-O-(3′-hydroxy)phytanyl-3-O-phytanyl-sn-glycerol) as a core lipid. In addition, two other ether phospholipids, usual archaetidylglycerol and archaetidylethanolamine, were also identified in the organism. The stereochemical structure of the unalkylated glycerophosphate of hydroxyarchaetidylglycerol and archaetidylglycerol was determined as sn-glycerol-3-phosphate by use of sn-glycerol-3-phosphate dehydrogenase. The stereochemical configuration of the glycerophosphoglycerol backbone of these lipids was a mirror image of that of diacylphosphatidylglycerol from the organisms of the domains Bacteria and Eucarya, and it was shared with extremely halophilic Archaea. These four phospholipids, in addition to five lipids that had already been reported, accounted for 88% of the total polar lipids of this organism.     

An improved method for the preparation of 1,2-isopropylidene-SN-glycerol

A simple and fast route for the preparation of 1,2-isopropylidene-sn-glycerol from D-mannitol in 45% yield is described. The value of optical rotation, [α]_D²⁰ + 15.2°, is higher than usual indicating considerable racemization for other procedures. Since 1,2-isopropylidene-sn-glycerol serves as general intermediate for the synthesis of glycerides and of phosphoglycerides these lipids contain substantial amounts of the isomer, for instance 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine may consist of up to 15% of 2,3-dipalmitoyl-sn-glycerol-1-phosphocholine in earlier preparations.     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Identification of sn-Glycerol-1-phosphate Dehydrogenase Activity from Genomic Information on a Hyperthermophilic Archaeon,Sulfolobus tokodaii Strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).