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.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.     

Fatty acid synthesis in Escherichia coli

1. Fatty acid formation by cells of a strain of Escherichia coli has been studied in the exponential, post-exponential and stationary phases of growth. 2. During the exponential phase of growth, the metabolic quotient (mmumoles of fatty acid synthesized/mg. dry wt. of cells/hr.) for each fatty acid in the extractable lipid was constant. 3. The newly synthesized fatty acid mixtures produced during this phase contained hexadecanoic acid (41%), hexadecenoic acid (31%), octadecenoic acid (21%) and the C(17)-cyclopropane acid, methylenehexadecanoic acid (4%). 4. As the proportion of newly synthesized material increased, changes in the fatty acid composition of the cells during this period were towards this constant composition. 5. Abrupt changes in fatty acid synthesis occurred when exponential growth ceased. 6. In media in which glycerol, or SO(4) (2-) or Mg(2+), was growth-limiting there was a small accumulation of C(17)-cyclopropane acid in cells growing in the post-exponential phase of growth. 7. Where either NH(4) (+) or PO(4) (3-) was growth-limiting and there were adequate supplies of glycerol, Mg(2+) and SO(4) (2-), there was a marked accumulation of C(17)-cyclopropane acid and C(19)-cyclopropane acid appeared. 8. Under appropriate conditions the metabolic quotient for C(17)-cyclopropane acid increased up to sevenfold at the end of exponential growth. Simultaneously the metabolic quotients of the other acids fell. 9. A mixture of glycerol, Mg(2+) and SO(4) (2-) stimulated cyclopropane acid formation in resting cells.     

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%.     

Osmotic regulation of PhoE porin synthesis in Escherichia coli.

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.     

Chromosome partition in Escherichia coli requires postreplication protein synthesis.

After inhibition of protein synthesis, the number of nuclear bodies (nucleoids) visible in cells of Escherichia coli B/rA corresponded closely to the number of completely replicated chromosomes. We calculated that nucleoid partition follows almost immediately after replication forks reach the chromosome terminus. We show that such a partition is dependent on protein synthesis and that this may reflect the requirement that cells must achieve a certain minimum length before partition (and subsequent cell division) can take place.     

DNA-directed in vitro synthesis of Escherichia coli beta-isopropylmalate dehydrogenase.

The in vitro synthesis of beta-isopropylmalate dehydrogenase (EC 1.1.1.85), an enzyme involved in leucine biosynthesis, has been obtained using as template DNA from the hybrid plasmid (pLC1) which contains the Escherichia coli leucine operon. Enzyme synthesis in vitro is stimulated about 2-fold by guanosine-5'-diphosphate-3'-diphosphate and inhibited about 60% by 2 X 10(-4) M L-leucine.     

Specificity and selectivity of diacylglycerolphosphate synthesis in Escherichia coli.

The glycerolphosphate and 1-acylglycerolphosphate acyltransferase systems Escherichia coli membranes show relatively low specificities for acylcoenzymes A when maximal velocities for the respective acyl-coenzymes A are compared. However, the selectivities for palmitate and oleate in the acylations of the 1- and 2-positions of glycerolphosphate moiety, respectively, are higher at lower concentrations of acceptors in the presence of an equimolar mixture of palmitoyl-CoA and oleoyl-CoA. More 1-palmitoyl-2-oleoyl-glycerolphosphate species and less other species were synthesized at lower concentrations of glycerolphosphate. The fatty acyl moiety at the 1-position of 1-acylglycerolphosphate did not influence significantly the specificity for acyl-coenzymes A of the 1-acylglycerolphosphate acyltransferase system. Thus, the acceptor concentrations being kept low in vivo and in vitro are important for the highly selective incorporations of saturated and unsaturated fatty acids into the 1- and 2-positions of diacylglycerolphosphate, respectively, in the presence of mixtures of saturated and unsaturated acyl-coenzymes A while these acyltransferase systems exhibit relatively low specificies for acyl-coenzymes A when the respective maximal velocities are compared.     

Induction of recA+-protein synthesis in Escherichia coli.

Summary Escherichia coli was infected with precA ⁺to determine the genetic and physiological factors controlling recA ⁺gene expression. When precA ⁺replication was prevented by superinfection immunity, recA ⁺protein synthesis was induced by UV radiation. The recA ⁺gene is negatively controlled by the lexA ⁺gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA ⁺protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA ⁺protein synthesis. Conversely positive control of recA ⁺gene expression requires recA ⁺protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA ⁺protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA ⁺protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA ⁺protein. lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA ⁺protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA ⁺and recA ⁺proteins normally combine to repress the recA ⁺gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA ⁺protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as repressor.     

Lipid synthesis during the Escherichia coli cell cycle.

Lipid synthesis was examined in Escherichia coli cells at different stage of cell division. Exponentially growing cells were pulse-labeled with appropriate isotopes for 0.1 generation time, inactivated, and separated by size on a sucrose gradient. An abrupt increase in the rate of lipid synthesis occurred which was coincident with the initiation of cross walls. In contrast, the rate of protein synthesis during this same interval remained constant, resulting in an increased lipid/protein ratio in dividing cells. No changes in the composition of phospholipid head groups, fatty acids, or phospholipid molecular species were observed in cells at different stages of division. The observed increase in the rate of lipid synthesis may reflect a means by which the activities of membrane-associated enzymes are modulated during cross wall formation.     

Control of glutaminase synthesis in Escherichia coli

Summary Glutaminase levels in E. coli B after growth on various media are presented. The results show that glutaminase synthesis is repressible and suggest glutamine as the corepressor.