Fatty acid synthesis in Escherichia coli is indirectly inhibited by phenethyl alcohol.

Experiments were performed to determine how phenethyl alcohol inhibits phospholipid synthesis in E. coli. At a nonbacteriostatic concentration, the drug reduces the rate of de novo fatty acid and phospholipid synthesis by 60 to 70%. The inhibition of fatty acid synthesis was found to be a secondary consequence of the inhibition of phospholipid synthesis. Phenethyl alcohol reduces the rate of incorporation of exogenous fatty acids into the phospholipids of a fatty acid auxotroph by 60%. These results indicate that this drug controls phospholipid synthesis beyond the level of fatty acid synthesis. Phenethyl alcohol inhibits the synthesis of phospholipids containing saturated fatty acids to a greater extent than it does the synthesis of phospholipids containing unsaturated fatty acids. It controls the synthesis of phospholipids containing saturated fatty acids at both the level of fatty acid synthesis and the level of incorporation of the saturated fatty acids into phospholipids. The synthesis of phospholipids containing unsaturated fatty acids is inhibited at the level of incorporation of the fatty acids into phospholipids.     

Phosphatidic acid synthesis in Escherichia coli

The kinetic properties of acyl-coenzyme A (CoA): l-alpha-glycerol-phosphate trans-acylase (EC 2.3.1.15) from Escherichia coli were studied. At 10 C, a temperature at which the reaction was proportional to time and enzyme concentration, the enzyme had an apparent K(m) of 60 mum for l-alpha-glycerol-phosphate. The curve describing the velocity of the reaction as a function of palmitoyl-CoA concentration was sigmoid but the plot of v(-1) versus [S](-3) gave a straight line. A K(m) of about 11 mum was calculated for palmitoyl-CoA. Adenosine triphosphate specifically inhibited the reaction, being a noncompetitive inhibitor in respect to l-alpha-glycerol phosphate. Inhibition only occurred with high concentrations of palmitoyl-CoA, and maximal inhibition was 60%.     

5-Aminolevulinic acid synthesis in Escherichia coli.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.     

Fatty acid composition of Escherichia coli cells and their viability in air

Experiments on E. coli used as a model have revealed that fatty-acid composition is one of the characteristics which determine the viability of bacteria in the air. The viability of microbial cells in the air has been shown to increase with the increase of the pool of cyclopropane acids and the palmitic acid/palmitoleic acid ratio in the cells, irrespective of their genotype and the phase of their growth.     

Fatty acid profile of Escherichia coli during the heat-shock response.

The possible changes in the fatty acid profile of Escharichia coli during heat-shock have been investigated. Bacteria growing in steady-state at 30 degrees C were subjected to an abrupt temperature upshift to 45 degrees C and held at the high temperature for various periods of time in order to elicit the heat-shock response. Fatty acid compositions of lipids extracted from samples taken at different times after the temperature upshift, as well as from cultures in steady-state at 30 and 45 degrees C, were determined by gas-chromatography. It has been found that the total unsaturates to total saturates ratio decreases gradually during heat-shock and that 30 min after the temperature jump, the reduction is equivalent to 57% of the difference between ratios corresponding to steady-state cultures at 30 and 45 degrees C. Consistent with this remodeling of lipid acyl chains, there is a decrease in the excimerization rate of the fluidity probe dipyrenylpropane incorporated into sonicated E. coli lipid extracts. Such modifications occur within the time-span of the heat-shock response, as judged from our previous measurements of the kinetics of change in heat-shock proteins induction ratio. Together, these results indicate that the control of membrane fluidity during the heat-shock response can be accounted for, at least in part, by an important change in the fatty acid composition of Escherichia coli lipids.     

Nucleic acid synthesis and nucleotide pools in purine-deficient Escherichia coli

1. The synthesis of nucleic acids and the content of purine nucleotides have been studied in selected purine-requiring strains of Escherichia coli including a purB(-) strain and a purB(-)guaA(-) strain. 2. When the exogenous purines can be converted into GTP but not into ATP, RNA is synthesized at the expense of intracellular ATP, ADP and AMP. 3. Net synthesis of RNA as measured by the incorporation of uracil can be correlated with the availability of GTP except when ATP falls to a very low concentration. 4. Nicotinamide nucleotides are not an important reservoir of adenine nucleotides for RNA synthesis.     

Fatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coli

Summary A short time after the immobilization of Escherichia coli in calcium alginate substantial modifications of the fatty acid patterns of the cells were observed. This effect could be related to lipid impurities in the commercial alginate product used, which could be taken up, at least in part by the microorganisms. The impurities were mainly free fatty acids but sterols were also detected. Immobilization of the cells in alginate material extracted by chloroform or ethanol decreased the tolerance of the cells to phenol as compared with cells immobilized in raw alginate. This effect was diminished if the immobilized cells were exogenously supplied with palmitic acid, which is the main constituent of the fatty acids extracted from alginate. These results indicate that not only fatty acids but also other ingredients of commercial alginate have physiological effects on cells entrapped in this gel material. Offprint requests to: H.-J. Rehm     

Fatty Acid Replacements in a Fatty Acid Auxotroph of Escherichia coli

Unsaturated fatty acids having structural features which are different from those of the monoenoic acids normally synthesized by Escherichia coli can serve as growth factors for an auxotroph requiring unsaturated fatty acids. These analogues were incorporated into the phospholipids, as shown by gas-liquid and thin-layer chromatographic analysis of the phospholipid fatty acid composition. Some of these fatty acids were cisDelta(5)- and cis-Delta(9)-tetradecenoic, cis-Delta(11)-eicosenoic, cis,cis-Delta(11,14)-eicosadienoic, cis,cis,cis-Delta(11,14,17)-eicosatrienoic, trans-Delta(9)- and trans-Delta(11)-octadecenoic acids. Although partial degradation of some of these analogues to shorter even-chain homologues occurred, chain elongation of the exogenous fatty acids was not detected. Trans-olefinic acids were utilized without stereochemical or positional isomerization. These studies provide a basis for exploring the properties of the fatty acids and phospholipids required for the formation, structure, and function of membranes.     

Gene controlling L-glutamic acid decarboxylase synthesis in Escherichia coli K-12

Genetically related Escherichia coli K-12 strains were found to differ widely in their l-glutamic acid decarboxylase (GAD) activity. This variation is due to differences in the amount of GAD produced by the different cultures, rather than to the appearance of altered enzymes differing in catalytic activity. A regulatory gene, gadR, which controls the amount of GAD was mapped on the E. coli K-12 chromosome. A strain with a lesion in the structural gene for GAD is described.