Nicotinic acid transport in Escherichia coli.

The uptake of nicotinic acid by Escherichia coli is dependent on the presence of the enzyme nicotinic acid phosphoribosyl transferase and a source of energy. Glucose concentrations between 0.1 and 0.5%, a temperature of 46 degrees C and an external concentration of 2.5 X 10(-5) were optimal conditions for nicotinic acid uptake. Saturation kinetics occur with a Km of 1.75 microM and a Vmax of 0.116 nmoles/min/mg dry weight. The intracellular molarity of the accumulated pyridine compounds is 44-fold that of the initial concentration. Inhibitors of respiration and anaerobiosis do not significantly inhibit uptake rate. However, an inhibitor of glycolysis, uncouplers of ATP production and sodium arsenate reduce vitamin transport. A mutant defective in ATPase does not accumulate exogenously supplied nicotinic acid when lactate is used as an energy source, although L-proline, the transport of which is independent of ATP production, is accumulated.

Isoniazid perturbation of the pyridine nucleotide cycle of Escherichia coli.

Isogenic strains of Escherichia coli, differing only in their sensitivity/resistance to isoniazid, were compared on the basis of intracellular accumulation of the intermediates of the pyridine nucleotide cycle and a closely related compound, nicotinamide adenine dinucleotide phosphate. Isoniazid treatment was bacteriostatic for the wild type organism and resulted in an exaggerated lag phase followed by a rapid logarithmic growth rate in the isoniazid-resistant strain. Isoniazid treatment of the wild type strain, even in the absence of growth, resulted in a temporal shift of peak pyridine accumulation from early lag phase to early logarithmic phase. The effect of mutation from isoniazid sensitivity to isoniazid resistance was accompanied by this same temporal shift of peak pyridine accumulation. In addition, mutation was accompanied by increases in the peak concentrations, as compared to wild type, of desamidonicotinamide adenine dinucleotide, nicotinamide, nicotinic acid and nicotinamide mononucleotide; the levels of nicotinic acid mononucleotide, nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide were decreased. Isoniazid treatment of the isoniazid-resistant strain, even in the absence of bacteriostasis, caused a decrease in the intracellular concentrations of nicotinic acid and nicotinamide and an increase in nicotinic acid mononucleotide. Isoniazid treatment of both the sensitive and resistant strains resulted in the loss of a peak ratio of NADP to NAD which normally occurred in the late logarithmic/early stationary phase of growth in untreated cultures. This denoted a loss of ability to shift from the generation of energy from glycolysis to generation of energy from the hexose monophosphate shunt.

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.

Modification of aspartate before its condensation with dihydroxyacetone phosphate during quinolinic acid formation in Escherichia coli.

A crude enzyme preparation from a nadA mutant of Escherichia coli was used to catalyze the conversion of [14C]aspartic acid into a precursor of quinolinic acid, a key intermediate in the biosynthesis of nicotinamide adenine dinucleotide.

Correlation of induced drug metabolism with titer of duck hepatitis virus in chickens.

While chickens infected with duck hepatitis virus showed no signs of clinical illness, their levels of hepatic cytochrome P-450 in response to phenobarbital induction and their microsomal aryl hydrocarbon hydroxylase activities in response to 3-methylcholanthrene induction were each found to correlate with the titer of virus recovered from the livers. These clear correlations indicate that avian hepatic drug metabolism is significantly modified during viral infection.

Studies on the modes of action of azaserine in Escherichia coli. Mechanism of resistance to azaserine.

Growth of wildtype Escherichia coli was inhibited by azaserine. There was an inverse relationship between the initial rate of uptake of phenylalanine and the azaserine concentration. Moderately azaserine-resistant mutants exhibited an initial rate that was similar to that of an aroP mutant, but highly azaserine-resistant mutants exhibited little, if any, uptake of phenylalanine. All of the azaserine-resistant organisms tested harboured a mutation in the aroP+ gene. However, resistance to the antibiotic was not due solely to this lesion.

A method for the rapid separation and characterization of biological pyridines.

Pyridine compounds of biological origin are separable by 2-dimensional descending paper chromatography. When assayed in situ for a tertiary or quaternary ring nitrogen, each compound gives a charactertistic reaction which is not predictable from the structural configuration. A cell-free extract of Escherichia coli, incubated in the presence of isoniazid, yields an ultraviolet light quenching spot with Rf values different from any tested standard. The compound is hypothesized to be a metabolite of either nicotinic acid or isonazid.

Detection of precursors of quinolinic acid in Escherichia coli.

A technique is described which allows the detection of precursors of quinolinic acid produced by Escherichia coli, independent of a bioassay. This is based on a double autoradiogram utilizing radioactive aspartic acid and fructose-1,6-diphosphate (as a source of dihydroxyacetone phosphate). Six radioactive spots are derived from aspartic acid and four are derived from fructose-1,6-diphosphate. The latter four spots correspond to four of the former spots in migration in each of two solvent systems. The significance of these data relative to genetic and enzymatic data is discussed.

Isolation of a metabolite capable of differentially supporting the growth of nicotinamide adenine dinucleotide auxotrophs of Escherichia coli.

A compound, isolated from the culture fluid of a nadC auxotroph of Escherichia coli grown in a minimal medium, supports the growth of both a nadA and a nadB mutant. This metabolite exhibits an ultraviolet light absorption spectrum and a mass spectrum, different from quinolinic acid. This compound may be the precursor of quinolinic acid, an intermediate in the biosynthesis of nicotinamide adenosine dinucleotide.

Protection of Escherichia coli from isoniazid inhibition.

Inhibition of Escherichia coli by isonicotinic acid hydrazide (isoniazid) is a function of the initial cell concentration, concentration of antibiotic, and chemical composition of the medium. An initial concentration of 5 x 10(5) cells/ml in a minimal medium is inhibited by 1 mM isoniazid. The E. coli cells are protected from this inhibitory effect by a high concentration of cells in the medium. Protection is also obtained from vitamin-free Casamino Acids, methionine, or choline plus homocystine. However, nicotinic acid, nicotinamide, and pyridoxamine are not able to reverse the effect of isoniazid. Colonies arising on minimal medium supplemented with isoniazid are not due to selection of resistant mutants, because this resistance is transitory and not passed on to the daughter cells. It is hypothesized that this transitory resistance is a manifestation of the cells' ability to transfer the methyl group of methionine to either isoniazid or accumulated nicotinic acid and/or nicotinamide.

Cross-feeding of Escherichia coli mutants defective in the biosynthesis of nicotinamide adenine dinucleotide.

Mutants of Escherichia coli defective in the biosynthesis of nicotinamide adenine dinucleotide (NAD) are able to grow in a Casamino Acids medium lacking NAD and its immediate precursors, nicotinic acid and nicotinamide. This property has allowed the development of a system to measure cross-feeding between a nadA and a nadB mutant. This system provides a means of isolating the intermediate, prequinolinic acid, as well as a biological assay for the compound. The nadB mutant feeds the nadA mutant, indicating that the nadA enzyme occurs first in the pathway and the nadB enzyme second. No cross-feeding was detected between nadA and nadC or between nadB and nadC.

Recognition of a gene involved in the regulation of nicotinamide adenine dinucleotide biosynthesis.

Mutations affecting the biosynthesis of quinolinic acid, a precursor of nicotinamide adenine dinucleotide (NAD) in Escherichia coli K-12, are either near min 17 (nadA mutants) or near min 49 on the chromosome. These nad mutants all exhibit a phenotypic requirement for NAD or one of its immediate precursors. The mutants with lesions near min 49 can be separated into two groups based on in vitro complementation analysis. One group (nadB) exhibits complementation with nadA mutants, whereas the other group fails to do so. The latter group is tentatively designated nadR based on its regulation of the unlinked nadA gene. The nadR gene maps adjacent to nadB between purI and tyrA.

Chromosomal location of the C gene involved in the biosynthesis of nicotinamide adenine dinucleotide in Escherichia coli K-12.

A gene involved in the synthesis of nicotinamide adenine dinucleotide has been found to be cotransducible with the genes involved in the utilization of arabinose (ara) and the biosynthesis of leucine (leu) and pantothenate (pan). Cotransduction frequency analysis places this nadC locus between leu and pan at approximately minute 1.5 on the genetic map of Escherichia coli. This gene codes for the enzyme, quinolate phosphoribosyl transferase, which catalyzes the conversion of quinolinic acid to nicotinic acid mononucleotide.

Identification of the purI locus in Escherichia coli K-12.

A genetic locus has been identified in Escherichia coli that is analogous to the purI locus in Salmonella.

Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escherichia coli K-12.

It is proposed that all mutants blocked in the de novo pathway of nicotinamide adenine dinucleotide biosynthesis be designated nad rather than nic. It is further suggested that mutants blocked in the pyridine nucleotide cycle be designated pnc. The nadB locus and a previously unidentified pur locus are cotransducible. These two loci have been mapped near minute 49 on the standard genetic map of Escherichia coli. The order of genes in that region is purC-guaB-purG-glyA-pur-nadB-tyrA-pheA.

Mode of nicotinamide adenine dinucleotide utilization by Escherichia coli.

Exogenous nicotinamide adenine dinucleotide is not utilized per se by Escherichia coli, but is converted to nicotinamide and thence to nicotinamide adenine dinucleotide via nicotinate.