The relationship of 4-hydroxybenzoic acid to lysine and methionine formation in Escherichia coli

1. A multiple aromatic mutant, Escherichia coli 156:53D2, required 4-hydroxybenzoic acid for rapid aerobic growth on a number of carbon sources. 2. In the absence of 4-hydroxybenzoic acid aerobic growth was stimulated by a mixture of lysine and methionine and by succinate. The influence of the amino acids is attributed to a sparing of succinyl-CoA. 3. Low activities of both alpha-oxoglutarate dehydrogenase and fumarate reductase were found in organisms grown aerobically without 4-hydroxybenzoate and consequently both mechanisms known for the formation of succinate were impaired. 4. The low fumarate-reductase activity in these organisms was due to repression of enzyme synthesis by aeration and not to enzyme inactivation. In contrast lactate dehydrogenase and ethanol dehydrogenase were induced. This is interpreted as the appearance of alternative routes of NADH oxidation when electron transfer to oxygen is impaired. 5. The activities of the other tricarboxylic acid-cycle enzymes tested were little influenced by 4-hydroxybenzoate deficiency, although anaerobiosis resulted in a fall in activity.     

Ubiquinone biosynthesis in Escherichia coli K-12. Accumulation of an octaprenol, farnesylfarnesylgeraniol, by a multiple aromatic auxotroph

Cell extracts of a multiple aromatic auxotroph of Escherichia coli K-12, strain AB2830, grown in the absence of precursors of the quinone rings of the ubiquinone and menaquinone molecules, converted 4-hydroxy[U-¹⁴C]benzoate into a mixture of 3-octaprenyl-4-hydroxybenzoate and 2-octaprenylphenol. An octaprenol, farnesylfarnesylgeraniol, was isolated from such cell extracts and characterized by n.m.r. and mass spectroscopy. Neither the octaprenol, nor polyprenylation of 4-hydroxy[U-¹⁴C]benzoate, could be detected in cell extracts of strain AB2830 grown in the presence of 0.1mm-4-hydroxybenzoate. It was concluded that, in the biosynthesis of ubiquinone, the polyprenyl side chain is added to 4-hydroxybenzoate as a C₄₀ unit, the active form of which is converted by cell extracts into farnesylfarnesylgeraniol. The multiple aromatic auxotroph, when grown in the absence of 4-hydroxybenzoate but in the presence of 4-aminobenzoate, converted the latter compound into 3-octaprenyl-4-aminobenzoate. This compound was isolated from whole cells and characterized by n.m.r. and mass spectroscopy.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli.

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFAts) grown at 28degrees C (PL28), and at 42degrees C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19degrees C for PL28 and at 43degrees C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42 degrees C) are in the state where the gel and liquid crystalline phases coexist.     

Growth in high osmotic medium of an unsaturated fatty acid auxotroph of Escherichia coli K-12

A temperature-sensitive mutant of Escherichia coli K-12, which is impaired in the biosynthesis of the unsaturated fatty acids at 42 C, continues to grow and remains viable in a medium with a high osmotic pressure. Under these growth conditions, 50% of the fatty acids of the parental strain and 80% of the fatty acids of the mutant strain are saturated.     

Biosynthesis of Ubiquinone in Escherichia coli K-12: Location of Genes Affecting the Metabolism of 3-Octaprenyl-4-hydroxybenzoic Acid and 2-Octaprenylphenol

Two genes (ubiB and ubiD) concerned with two successive reactions in ubiquinone biosynthesis in Escherichia coli were mapped and found to be closely linked. Mutant strains of E. coli carrying the ubiB(-) and ubiD(-) alleles were shown to accumulate 2-octaprenylphenol and 3-octaprenyl-4-hydroxybenzoic acid, respectively. These compounds were isolated and identified by using nuclear magnetic resonance and mass and infrared spectroscopy. Cell extracts from the mutant strain carrying the ubiD(-) allele lack 3-octaprenyl-4-hydroxybenzoate decarboxylase activity.     

Structural specificity of the spermidine requirement of an Escherichia coli auxotroph.

A homologous series of spermidine analogs was synthesized with the general structure NH3+ (CH2)nNH2+(CH2)3NH3+, where spermidine has n = 4. The influence of these compounds on growth and on the syntheses of protein and messenger ribonucleic acid was examined in a spermidine auxotroph of Escherichia coli. All of the homologs tested were taken up by the cells to an intracellular level equivalent to the level of spermidine which gives optimal growth. With increasing chain length of the homologs, there was reduced ability to stimulate growth. The homologs with n = 7 and n = 8 were essentially inactive. A similar specificity was observed when the ability of the homologs to restore the rates of protein and messenger ribonucleic acid chain elongation was compared to that of spermidine. These results suggest that a definite spatial arrangement of the amino groups of spermidine is required for productive interaction at its intracellular site(s) of action.     

Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb⁻ phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery.     

Biophysical properties of a major membrane phospholipid, dielaidoylphosphatidylethanolamine, found in an Escherichia coli fatty acid auxotroph.

Dielaidoylphosphatidylethanolamine, a principal lipid component of membranes of Escherichia coli fatty acid auxotrophs enriched in elaidic acid, has been studied by paramagnetic resonance, fluorescence, and calorimetric methods. EPR measurements with perdeutero-di-tert-butylnitroxide and 2,2,6,6-tetramethyl piperidine-1-oxyl indicate that, when dispersed in aqueous media, this phospholipid undergoes an abrupt order leads to disorder transition at 37.5 degrees C and 36.5 C, respectively. A similar transition temperature is suggested by experiments with 9-doxyl-dimyristoylphosphatidylethanolamine (DEPE). cis- and trans-Parinaric-acid fluorescence polarization measurements indicate that the midpoint of this transition occurs at 34.0 degrees C and 35.5 degrees C, respectively. Differential scanning calorimetry of DEPE revealed a single, sharp endotherm at 38.5 degrees C with increasing temperature; two exotherms of similar magnitude were observed at 36.5 degrees C and 34.5 degrees C upon cooling. This double transition was not observed by any of the other methods. From these results we conclude that the major structural transition at 30-31 degrees C observed previously with 5-, 12-, and 16-doxyl stearate in intact E. coli membranes is due to the DEPE present (Morrisett, J.D., Pownall, H.J., Plumlee, R.T., Smith, L.C., Zehner, Z.E., Esfahani, M., and Wakil, S.J. (1975) J. Biol. Chem. 250, 6969-6976).     

A bioluminescent Escherichia coli auxotroph for use in an in vitro lysine availability assay

Microbiological methods have been used to determine the amino acid availability of a variety of animal feed and human food protein sources. Growth of Escherichia coli auxotrophs have been shown to yield a consistent linear response to lysine concentration when compared to chemical measures. Extent of total growth of E. coli lysine mutant (American Type Culture Collection #23812) when measured as optical density (OD) displays a lysine-dependent growth response that can be used to estimate lysine in feed proteins. However, typical OD-based growth studies for amino acid quantitation using the mutant may require anywhere from 12 to over 40 h. To develop an improved rapid method for lysine quantitation in protein sources, the plasmid pJHD500 carrying genes that encode for expression of bioluminescence and ampicillin resistance was transformed into the E. coli mutant by electroporation (set at 1.80 kV). The luminescence measured during early exponential growth allowed detectable differentiation of lysine concentration in the media in 4 h. When the luminescence method was compared with the conventional optical density lysine growth assay, the correlation coefficient was 0.989. Lysine availability valued for enzymatically hydrolyzed protein sources were comparable with availability measures using animal methods for lysine availability. This research shows potential applications for more rapid quantitative measurement of bioavailable lysine.     

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.     

Influence of unsaturated fatty acid membrane component on sensitivity of an Escherichia coli fatty acid auxotroph to conditions of nutrient depletion

The unsaturated fatty acid auxotroph Escherichia coli AK7 was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18:3) to vary the degree of unsaturation of cell membrane lipids. The susceptibility of oleic acid- and linolenic acid-grown cells to starvation at 37 degrees C in 154 mM NaCl was compared following the decline in the number of CFU by plating the cells on agar medium. The decline in CFU was faster for linolenic acid-than for oleic acid-grown cells, but it was not indicative of cell death, since culturable CFU was recovered after respirable substrate was added to the starved cell suspension. Cell envelope microviscosity (determined by fluorescence polarization) of oleic acid- and linolenic acid-grown cells was equal in the presence of a respirable substrate, but in its absence the microviscosity of linolenic acid-grown cells was lower than that of oleic acid-grown cells. The results suggest that cell envelope microviscosity is an important factor in determining the sensitivity of E. coli to conditions of nutrient depletion.     

Influence of unsaturated fatty acid membrane component on sensitivity of an Escherichia coli fatty acid auxotroph to conditions of nutrient depletion.

The unsaturated fatty acid auxotroph Escherichia coli AK7 was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18:3) to vary the degree of unsaturation of cell membrane lipids. The susceptibility of oleic acid- and linolenic acid-grown cells to starvation at 37 degrees C in 154 mM NaCl was compared following the decline in the number of CFU by plating the cells on agar medium. The decline in CFU was faster for linolenic acid-than for oleic acid-grown cells, but it was not indicative of cell death, since culturable CFU was recovered after respirable substrate was added to the starved cell suspension. Cell envelope microviscosity (determined by fluorescence polarization) of oleic acid- and linolenic acid-grown cells was equal in the presence of a respirable substrate, but in its absence the microviscosity of linolenic acid-grown cells was lower than that of oleic acid-grown cells. The results suggest that cell envelope microviscosity is an important factor in determining the sensitivity of E. coli to conditions of nutrient depletion.     

Function of Ubiquinone in Escherichia coli: a Mutant Strain Forming a Low Level of Ubiquinone

A ubiquinone-deficient mutant of Escherichia coli K-12 forming 20% of the normal amount of ubiquinone was compared with a normal strain. This lowered concentration of ubiquinone is still four times the concentration of cytochrome b(1). The mutant strain grew more slowly than the normal strain on a minimal medium with glucose as sole source of carbon and gave a lower aerobic growth yield than the normal strain. The reduced nicotinamide adenine dinucleotide (NADH) oxidase rate in membranes from the mutant strain was 40% of the oxidase rate in membranes from the normal strain, and the percentage reduction of cytochrome b(1) in the aerobic steady state, with NADH as substrate, was increased in membranes from the mutant strain. It is concluded that ubiquinone is required for maximum oxidase activity at the relatively high concentration (27 times that of cytochrome b(1)) found in normal cells. The results are discussed in relation to a scheme previously advanced for ubiquinone function in E. coli.     

Heterogeneous amino acid transport rate changes in an E. coli unsaturated fatty acid auxotroph.

Amino acid transport rates in an $\text{E. coli}$ unsaturated fatty acid auxotroph were non-uniformly affected by enrichment of membrane lipids in various unsaturated fatty acids. Proline and threonine transport rates were depressed much more than lysine and asparagine rates by trans unsaturated acids. Myristoleate and linolenate enrichment also produced non-uniform but lesser rate reductions. Although changes in the relative numoer of effective transport catalysts could account for these findings, comparisons of proline and lysine transport rates over a broad temperature range indicated that non-uniform alterations in transport catalyst reaction rates account at least partly for the activity changes associated with membrane lipid alterations.     

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.     

Biochemical and Genetic Studies on Ubiquinone Biosynthesis in Escherichia coli K-12: 4-Hydroxybenzoate Octaprenyltransferase

Three ubiquinone-deficient mutants of Escherichia coli unable to convert 4-hydroxybenzoate into 3-octaprenyl-4-hydroxybenzoate were isolated and examined. The results of genetic analysis suggest that each of the mutants carries a mutation in a gene designated ubiA which can be represented at minute 79 on the E. coli chromosome map. The conversion of 4-hydroxybenzoate into 3-octaprenyl-4-hydroxybenzoate, catalyzed by 4-hydroxybenzoate octaprenyltransferase, was studied with a strain of E. coli that is blocked in the common pathway of aromatic biosynthesis and consequently accumulates the precursor of the side chain of ubiquinone. Both the side-chain precursor and 4-hydroxybenzoate octaprenyltransferase were shown to be membrane-bound. The enzyme required Mg(2+) for optimal activity. The ubiA(-) mutants were found to lack 4-hydroxybenozate octaprenyltransferase activity, which suggested that the ubiA gene is the structural gene coding for this enzyme.