Biomedical subjects

C R Raetz

Publications and source records attributed to C R Raetz.

At least 127 records · Page 7Linked to original sources

PubMed1987

Biosynthesis of lipid A precursors in Escherichia coli. A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X.

Certain phosphatidylglycerol-deficient mutants of Escherichia coli accumulate two fatty acylated monosaccharides related to lipid A biosynthesis that have been identified as 2,3-diacylglucosamine 1-phosphate (lipid X) and triacylglucosamine 1-phosphate (lipid Y) (Raetz, C. R. H. (1984) Rev. Infect. Dis. 6, 463-472). Lipid Y has the same structure as lipid X, except that it bears an additional palmitoyl moiety, esterified to the 3-OH of the N-linked R-3-hydroxymyristoyl residue. We now describe a membrane-associated system for the enzymatic conversion of lipid X to lipid Y. Removal of glycerophospholipids form such membranes by washing with cold ethanol abolishes the activity. The system can be reactivated by the addition of exogenous phospholipids dispersed as mixed micelles with Triton X-100. When reconstituted in this manner, the formation of lipid Y is strictly dependent upon a glycerophospholipid donor bearing a palmitoyl residue in the sn-1 position. The enzyme system does not utilize palmitoyl coenzyme A or palmitoyl acyl carrier protein. It does not catalyze efficient transfer of fatty acids differing from palmitate by only one carbon atom. In contrast, the enzyme has relatively little specificity for the polar headgroup of the phospholipid donor, and it also appears to utilize a disaccharide precursor of lipid A as an alternative palmitoyl acceptor. Since the in vitro synthesis of lipid Y proceeds with a high yield, we have isolated the product and verified its structure by 1H NMR spectroscopy and mass spectrometry. The transesterification reaction that converts lipid X to lipid Y may be a model for the enzymatic synthesis of other acyloxyacyl structures, known to occur in mature lipid A.

Acyltransferases↗

PubMed1987

The biosynthesis of gram-negative endotoxin. A novel kinase in Escherichia coli membranes that incorporates the 4'-phosphate of lipid A.

Extracts of Escherichia coli contain an enzyme that generates the beta,1----6 linkage of lipid A from fatty-acylated monosaccharide precursors, according to the reaction: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). We now describe a membrane-bound kinase that phosphorylates the 4'-position of the above tetraacyldisaccharide 1-phosphate product. The lipid A 4'-kinase is distinct from the diglyceride kinase of E. coli. When crude membrane preparations are employed, several nucleoside triphosphates are able to support the phosphorylation of the tetraacyldisaccharide 1-phosphate, but ATP is the most efficient. The 4'-kinase requires Mg2+ and is stimulated by phospholipids, especially cardiolipin. Under optimal conditions the specific activity in crude extracts is 0.5 nmol/min/mg. The enzyme is rapidly inactivated by preincubation in the presence of detergents, such as Nonidet P-40 or octylglucoside, but phosphoenolpyruvate and glycerol stabilize the enzyme. The product generated in vitro has been characterized by fast atom bombardment mass spectrometry and by 1H and 31P NMR spectroscopy. Those analyses confirm that the 4' hydroxyl is the site of phosphorylation. The 4'-kinase reported here is likely to represent a key step in the de novo biosynthesis of lipid A.

Adenosine Triphosphate↗

PubMed1987

Lipid X ameliorates pulmonary hypertension and protects sheep from death due to endotoxin.

Lipid X (2,3-diacylglucosamine-1-phosphate) is a novel monosaccharide precursor of lipid A that has some of the physiologic activities of endotoxin but little toxicity. To determine whether lipid X would interfere with the toxic effects of endotoxin, we pretreated sheep with either 100 or 200 micrograms of lipid X per kg of body weight and then challenged them with a potentially fatal dose of Escherichia coli endotoxin (20 micrograms/kg). Twenty-one sheep underwent pulmonary artery catheterization and were monitored for changes in pulmonary artery pressure, temperature, pH, partial O2 pressure, partial CO2 pressure, blood pressure, and cell counts over 7 h. Overall mortality for control animals was 37% versus 5.3% for pretreated animals. None of the 13 animals pretreated with 100 micrograms of lipid X per kg died. These differences in survival were significant (P less than 0.05). Animals pretreated with 100 micrograms of lipid X per kg had significantly lower pulmonary artery pressure during both phases 1 and 2 of endotoxin-induced pulmonary artery hypertension. A higher dose of lipid X, 200 micrograms/kg, produced pulmonary hypertension. Perhaps because lipid X is a subunit of lipid A, lipid X shows a partial pyrogenic effect while also decreasing the pyrogenic activity of complete lipopolysaccharide (LPS). Lipid X did not prevent endotoxin-induced neutropenia or moderate hypotension in response to LPS. Lipid X is a potential prototype compound for a new type of chemotherapy directed at blocking the harmful effects of LPS during bacterial septicemia.

Animals↗

PubMed1987

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.

Acyltransferases↗

PubMed1987

Pulmonary pressor responses in sheep to chemically defined precursors of E. coli endotoxin.

The toxicity of various monosaccharide and disaccharide endotoxin precursors has now been studied in sheep. We measured the early pulmonary arterial pressure responses after injections of the monosaccharides lipid X (2,3-diacylglucosamine 1-phosphate) and MAGP (2-monoacylglucosamine 1-phosphate), of the tetraacyl disaccharide diphosphate precursor of lipid A, IV-A (Federation Proc. 43: 1567, 1984), and of Escherichia coli bacterial endotoxin (lipopolysaccharide). We also measured the response of lipid X after prior administration of indomethacin and MAGP. Lipid X, at a total cumulative dose of 40 micrograms/kg, produced an immediate, but transient dose-dependent pulmonary arterial vasoconstrictive response. MAGP, at a total dose of 40 micrograms/kg, had no pulmonary pressure activity but did increase extravascular lung water and produce some histological changes in the lung. Disaccharide precursor IV-A, at a total dose of 40 micrograms/kg, produced an immediate dose-dependent pulmonary arterial vasoconstrictive response that was prolonged for greater than 2 h. E. coli endotoxin caused a delayed (15-min) increase in the pulmonary arterial pressure but one that also persisted for greater than 2 h. Prior administration of indomethacin blocked the pulmonary pressor activity of lipid X, whereas prior administration of MAGP increased both the magnitude and the duration of the pulmonary pressure response of lipid X. We conclude that the initial pulmonary hypertension seen after lipid X injection may involve cyclooxygenase-dependent formation of prostaglandins and that the genesis of this pulmonary pressor activity is at least in part dependent on the ester-linked hydroxymyristoyl moiety at position 3 of the lipid X molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly.

A rapid autoradiographic screening procedure has been developed for identifying Chinese hamster ovary cell mutants defective in the peroxisomal enzyme dihydroxyacetonephosphate (DHAP) acyltransferase. Ten mutants were found among 60,000 colonies grown from a stock of mutagen-treated cells, and 3 have been characterized with respect to their enzymology and phospholipid biosynthesis. All three contain 3% (or less) of the parental DHAP acyltransferase activity measured at pH 5.5, the optimum for the peroxisomal enzyme. When measured at pH 7.4, all three contained 70-85% of the wild-type activity, but it was sensitive to N-ethylmaleimide. Glycerol-3-phosphate acyltransferase activities were identical in mutant and parent strains. Two other peroxisomal enzymes, alkyl-DHAP synthase and particulate catalase, were also reduced by factors of 5-10 in all three mutants, suggesting that these strains are deficient in some aspect of peroxisome assembly, possibly like cells from patients with Zellweger syndrome. Short-term and long-term labeling with 32Pi revealed that these mutants are grossly deficient in the de novo synthesis and content of plasmalogens. In parental cells the plasmalogen form of phosphatidylethanolamine constitutes 7.1% of the total phospholipid, but it is reduced to 0.7% in the mutants. This decrease is accompanied by a compensatory increase in the diacyl form of phosphatidylethanolamine. The results presented here support the view that there are two DHAP acyltransferases in animal cells and that the peroxisome is essential for the biosynthesis of plasmalogens.

Acyltransferases↗

PubMed1986

Protection of mice against lethal endotoxemia by a lipid A precursor.

Lipid X, the major biosynthetic precursor of lipid A, has recently been described. Although lipid X is a mitogen and coagulates the Limulus amebocyte lysate, we found that it is not lethal for mice, even when given in large doses (2 X 10(6) micrograms/kg). Furthermore, lipid X was found to give partial protection against a 100% lethal dose of endotoxin, even if the lipid X was given as late as 6 h after endotoxin challenge.

Animals↗

PubMed1986

Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli.

Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide. Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase. Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene. We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L. Clarke and J. Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E. coli chromosome. Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase. Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.

Acyltransferases↗

PubMed1986

Molecular genetics of membrane phospholipid synthesis.

I have attempted to illustrate the genetic and biochemical complexity of membrane-lipid synthesis by focusing, primarily, on E. coli. The use of molecular genetics to probe membrane lipids is relatively new. Many important questions of phospholipid biochemistry remain unanswered. In the coming years our growing knowledge of the molecular genetics of phospholipids must be applied to the solution of the following problems: How does a cell regulate its total phospholipid content in relationship to macromolecules, especially membrane proteins, cell wall components, and nucleic acids? Why do E. coli and Caulobacter behave differently in this respect? How does a cell regulate its characteristic ratios of polar headgroups and fatty acyl chains? Why does overproduction of phosphatidylserine synthase have no effect on phospholipid composition? How is lipid topography established, both in terms of intramembrane movement (flip-flop) and intermembrane movement? Are there transport systems (flippases) for short-chain diacylglycerophospholipids in E. coli, as in mammalian microsomes, and can flippase mutants be isolated? What are the functions of the many individual phospholipid species? Does E. coli have a functional equivalent of the mammalian phosphatidylinositol cycle? A complete set of phospholipid mutants, together with phenotypic suppressors, should help to answer these questions by allowing selective perturbations in vivo and physiological studies of associated phenotypes. In addition, molecular cloning is already providing access to large quantities of the lipid gene products, opening the door to biophysical and chemical studies of lipid-protein interactions. A unique feature of genetics, as applied to complex biochemical or physiological systems, is the high frequency of unanticipated discoveries that accompany the characterization of new mutants. In our work, this is best illustrated by the analysis of phosphatidylglycerol-deficient mutants of E. coli, which provided the clue (i.e. lipid X) that permitted the elucidation of lipid A biosynthesis. The interconnection of metabolic pathways and important control mechanisms are often revealed by the study of mutants. In the case of E. coli it is best to consider the many lipids and proteins of the envelope as a whole. Considering how few mutant alleles are available for the lipid genes of E. coli, it will be important to create many more genetic lesions in order to gain a full understanding of regulation and function.

Animals↗

PubMed1985

Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium.

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.

Glycolipids↗

PubMed1985

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance.

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.

Acylation↗

PubMed1985

The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli.

The Gram-negative bacterium Escherichia coli has previously been shown to utilize two unique glucosamine (GlcN)-derived phospholipids in the biosynthesis of lipid A disaccharides (Bulawa, C.E., and Raetz, C. R.H. (1984) J. Biol. Chem. 259, 4846-4851; Ray, B. L., Painter, G.L., and Raetz, C.R.H. (1984) J. Biol. Chem. 259, 4852-4859. We now present evidence that these compounds, UDP-2,3-diacyl-GlcN and 2,3-diacyl-GlcN-1-phosphate (2,3-diacyl-GlcN-1-P), are generated in extracts of E. coli by fatty acylation of UDP-GlcNAc. The initial reaction is an O-acylation of the glucosamine ring, presumably of the 3-OH group, with (R)-beta-hydroxymyristate, followed by removal of the acetyl moiety, and further fatty acylation of the N atom with (R)-beta-hydroxymyristate to yield UDP-2,3-diacyl-GlcN. Hydrolysis of the pyrophosphate bridge in this molecule gives 2,3-diacyl-GlcN-1-P + UMP. In vivo pulse labeling with 32Pi supports this postulated pathway, since UDP-2,3-diacyl-GlcN is labeled prior to 2,3-diacyl-GlcN-1-P. UDP-glucosamine is inactive as a substrate in the initial acylation reaction. These acylations show an absolute specificity for fatty acyl moieties activated with acyl carrier protein. No reaction is detected with fatty acyl-CoA or free fatty acid. The fatty acylation of sugar nucleotides has not been reported previously in E. coli or any other organism.

Acylation↗

PubMed1985

Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli.

The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J. Biol. Chem. 260, 12084-12091). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map.

Amino Acid Sequence↗

PubMed1985

Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli.

The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology. The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. Apparent physical effects of EDTA on the enzyme were also utilized in the purification. The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C. P., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 12078-12083). The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein. The substrate specificity of the enzyme is also investigated. This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms.

Amino Acids↗

PubMed1985

Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli.

Previous work from this laboratory had demonstrated that CDP-diglyceride hydrolase of Escherichia coli is encoded by the cdh gene that maps near minute 88 (Bulawa, C. E., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 11257-11264). We now report the construction of hybrid plasmids and the sequencing of a 1,243-base pair insert carrying cdh. The further construction of BAL31 deletions of this insert, in conjunction with maxicell experiments and in vitro enzyme assay, has led to the identification of a 756-base pair coding sequence for the cdh polypeptide. The molecular weight of the primary translation product deduced from the DNA sequence of the cdh gene is 28,450, in agreement with maxicell experiments. Parallel purification of the enzyme from extracts of wild-type and overproducing strains confirms the presence of a 27-kDa polypeptide in the overproducer, as judged by polyacrylamide gel electrophoresis of the most purified fractions. Inspection of the DNA sequence reveals a very hydrophobic N-terminal domain that may be either a signal peptide or a special region, anchoring the hydrolase to the membrane. In contrast to the CDP-diglyceride synthetase, the overall amino acid composition of the CDP-diglyceride hydrolase is not extraordinarily hydrophobic. Although both CDP-diglyceride synthetase and CDP-diglyceride hydrolase can transfer the CMP moiety of CDP-diglyceride to a suitable acceptor, the primary structures and mechanisms of action of these two enzymes are very different.

Amino Acid Sequence↗

PubMed1985

Macrophage activation by monosaccharide precursors of Escherichia coli lipid A.

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate two novel glycolipids, designated X and Y. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3, and lipid Y has the same structure as X, except for the additional presence of a palmitoyl moiety on the N-linked beta-hydroxymyristate. We have examined the activities of X, Y, and several related compounds as activators of macrophages. Both X and Y induce morphological changes (spreading), prostaglandin E2 synthesis, and killing of tumor cells by mouse peritoneal macrophages in vitro, properties with which lipopolysaccharide and lipid A are also endowed. Both glycolipids have similar effects on the macrophage-like mouse cell line J774.1. Selective removal from lipid X of either the ester-linked beta-hydroxymyristate at position 3 or the phosphate at position 1 abolishes activity. Our results show that the monosaccharides X and Y retain some of the properties of intact lipopolysaccharide and lipid A with respect to macrophage activation. Because the structures of X and Y are defined, our findings should facilitate the elucidation of the molecular mechanism of macrophage activation by lipid A.

Animals↗

PubMed1985

Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase.

Phosphotyrosine-Sepharose 4B was synthesized and used to purify L-cell alkaline phosphatase. Antibodies to this enzyme interacted with the alkaline phosphatase of strains A-1-2 and A-3-3, mutants that express the enzyme constitutively. This and thermal stability studies suggest that these mutants contain the same alkaline phosphatase isozyme as their parent strain.

Alkaline Phosphatase↗

PubMed1984

Escherichia coli membrane vesicles with elevated phosphatidic acid levels. A detergent-free system for in vitro phospholipid synthesis.

A new method for studying phospholipid biosynthesis in Escherichia coli is described. The method makes use of the previously reported observation that E. coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B. and Raetz, C.R.H. (1982) J. Biol. Chem. 257, 389-394). We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells. Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis. Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane. We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups. Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays. This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane.

Cell Membrane↗