Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action.

The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]acetate labeling of fatty acids under these conditions. In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis. By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth. Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures. Analysis of extracts of [35S]methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid. Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli thioesterase I or E. coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect. Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).     

Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production

The influence of heat adaptation (growth at 42 and 45 degrees C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57 degrees C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1omega7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.     

Escherichia coli mutants defective in membrane phospholipid synthesis: binding and metabolism of 1-oleoylglycerol 3-phosphate by a plsB deep rough mutant.

Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's.     

The change in membrane phospholipid composition influences protein secretion and cell envelope biogenesis in Escherichia coli

Secretion of periplasmic alkaline phosphatase (PhoA) encoded by the gene constituent of plasmids and the peculiar properties of cell envelope biogenesis in Escherichia coli strains with controlled synthesis of individual membrane phospholipids have been studied. Alkaline phosphatase secretion across the cytoplasmic membrane declines, while secretion into the culture medium intensifies under changed metabolism. The composition of anionic membrane phospholipids changes due to inactivation of the pgsA gene or regulation of its expression by environmental factor, as well as in the absence of the pssA gene which is responsible for the synthesis of the precursor for zwitter-ionic phospholipid — phosphatidylethanolamine. This correlates with intensified secretion of exopolysaccharides and lower content of lipopolysaccharide and lipoprotein which are responsible for barrier properties of the outer membrane. The results suggest a possible coupling of protein secretion with biogenesis of cell envelope components at a level of phospholipid metabolism.     

Lipid and lipopolysaccharide composition of Escherichia coli surface-altered mutants selected for resistance to levallorphan, tetracaine, and polymyxin.

Certain mutants of Escherichia coli with an altered permeability barrier have an essentially normal lipopolysaccharide, fatty acid, and phospholipid content, with a slight increase in the membrane protein:lipid ratio. The phospholipid metabolism of the lev and tec strains shows an abnormal response to growth in the selective agents levallorphan and tetracaine, respectively.     

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.     

Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli.

The penicillin tolerance exhibited by amino acid-deprived Escherichia coli has been previously proposed to be a consequence of the stringent response. Evidence indicating that penicillin tolerance is directly attributable to guanosine 3',5'-bispyrophosphate (ppGpp) overproduction and not to some other effect of amino acid deprivation is now presented. Accumulation of ppGpp in the absence of amino acid deprivation was achieved by the controlled overexpression of the cloned relA gene, which encodes ppGpp synthetase I. The overproduction of ppGpp resulted in the inhibition of both peptidoglycan and phospholipid synthesis and in penicillin tolerance. The minimum concentration of ppGpp required to establish these phenomena was determined to be 870 pmol per mg (dry weight) of cells. This represented about 70% of the maximum level of ppGpp accumulated during the stringent response. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis (R. J. Health, S. Jackowski, and C. O. Rock, J. Biol. Chem. 269:26584-26590, 1994). The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This result supports previous observations indicating that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly, treatment with beta-lactam antibiotics by itself induced ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.     

Outer Membrane Proteins of Escherichia coli IV. Differences in Outer Membrane Proteins Due To Strain and Cultural Differences

When the 42,000-dalton major outer membrane protein of Escherichia coli O111 is examined on alkaline polyacrylamide gels containing sodium dodecyl sulfate, it is resolved into three distinct bands designated as proteins 1, 2, and 3. Band 3 consists of two distinct polypeptides, proteins 3a and 3b. E. coli K-12 does not make any protein 2, but makes proteins similar to 1, 3a, and 3b as indicated by comparison of cyanogen bromide peptide patterns. Several Shigella species and most other strains of E. coli resemble E. coli K-12 in that they lack protein 2, whereas Salmonella typhimurium is more similar to E. coli O111. In addition to these species and strain differences, cultural differences resulted in differences in the outer membrane protein profiles. Under conditions of catabolite repression, the level of protein 2 in E. coli O111 decreased while the level of protein 1 increased. An enterotoxin-producing strain similar to E. coli O111 produced no protein 1 and an elevated level of protein 2 under conditions of low catabolite repression. The levels of proteins 1 and 3 are also different in different phases of the growth curve, with protein 1 being the major species in the exponential-phase cells and protein 3 being the major species in stationary-phase cells. A multiply phage-resistant mutant of E. coli K-12 with no obvious cell wall defects produced no protein 1 or 2, but made increased amounts of protein 3. Thus, the major outer membrane proteins of E. coli and related species may vary considerably without affecting outer membrane integrity.     

Metabolic regulations and biological functions of phospholipids in Escherichia coli.

Extensive genetic and biochemical studies in the last two decades have elucidated almost completely the framework of synthesis and turnover of quantitatively major phospholipids in E. coli. The knowledge thus accumulated has allowed to formulate a novel working model that assumes sophisticated regulatory mechanisms in E. coli to achieve the optimal phospholipid composition and content in the membranes. E. coli also appears to possess the ability to adapt phospholipid synthesis to various cellular conditions. Understanding of the functional aspects of E. coli phospholipids is now advancing significantly and it will soon be able to explain many of the hitherto unclear cell's activities on the molecular basis. Phosphatidylglycerol is believed to play the central role both in metabolism and functions of phospholipids in E. coli. The results obtained with E. coli should undoubtedly be helpful in the study of more complicated phospholipid metabolism and functions in higher organisms.     

The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

Changes in the composition of anionic membrane phospholipids influence the protein secretion and biogenesis of cell envelope in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD 102 and HDL11 with controlled synthesis of anionic phospholipids, phosphatidylglycerol, and cardiolipin. Inactivation of the pgsA gene encoding the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. A conforming increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of lipopolysaccharide and lipopolyprotein of the outer membrane that determine the membrane barrier properties. The results obtained testify that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between the protein secretion and biogenesis of cell envelope components.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.     

The major phospholipid of Escherichia coli, phosphatidylethanolamine, is required for efficient production and secretion of alkaline phosphatase

The major phospholipid of the Escherichia coli membranes--the zwitterion phosphatidylethanolamine (PE)--is the only phospholipid involved in the formation of non-bilayer structure of membrane lipids, which is supposed to be necessary for efficient translocation of secreted proteins across the cytoplasmic membrane. The effect of PE on the production and secretion of alkaline phosphatase has been studied in this work using the mutant strain E. coli AD93, which is unable to synthesize PE. It was shown that this phospholipid is required for the efficient production and secretion of alkaline phosphatase. The anionic phospholipid cardiolipin in combination with divalent cations Mg2+ functionally replaces PE in these processes, participating in the regulation of lipid polymorphism.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

Lipid metabolism in human endothelial cells

Although lipids are largely involved in cardiovascular physiopathology, the lipid metabolism in endothelial cells remains largely unknown. Human umbilical vein endothelial cells (HUVECs) were used to investigate the metabolism of complex lipids. The membrane phospholipid homeostasis results from both de novo synthesis and remodelling that ensures the fine tuning of the phospholipid fatty acid composition. Using [(3)H]-glycerol and phosphoderivatives we showed the efficiency of glycerolipid synthesis from glycerol (0.9 nmol h(-1) mg proteins(-1)), but not from its phosphorylated form suggesting the requirement of a functional glycerol kinase in HUVECs. Conversely, the synthesis of triacylglycerols was very low (less than 5% of phospholipid synthesis). The incorporation rate of fatty acids into phospholipids showed that there is a specific fate for each fatty acid in respect to its chain length and saturation level. Moreover in steady state condition, increasing the long chain omega3 polyunsaturated fatty acids in the medium resulted in an increased polyunsaturated/saturated ratio in phospholipids (from 0.42 to 0.63). [(14)C]O(2) was produced form either [(14)C]-glucose or [(14)C]-palmitate indicating the functionality of the oxidation pathways, although beta-oxidation was less efficient than glucose oxidation. The endothelial cell lipid metabolism involves conventional pathways, with functional rates largely slower than in hepatocytes or in cardiomyocytes.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.