Agnete Munch-Petersen

Studies on the acid-soluble nucleotide pool in thymine-requiring mutants of Escherichia coli during thymine starvation: II. Changes in the amounts of deoxycytidine triphosphate and deoxyadenosine triphosphate in Escherchia coli 15 T-A-U-

Abstract By use of two-dimensional thin-layer chromatography on anion-exchange cellulose it has been possible to separate the normally occurring ribo- and deoxyribonucleoside triphosphates. With this technique the amounts of the different triphosphates were determined in extracts of Escherichia coli 15 T-A-U- labelled with [ 32 P]orthophosphate under different conditions of thymine starvation. A 4–5-fold increase in deATP content occurs immediately after thymine removal. This increase is independent of concomitant protein synthesis. After 30–60 min no further increase is observed. In contrast, the deCTP content remains constant for 30 min after removal of thymine and then rises steeply reaching a 10–15-fold value after 90 min of thymine starvation. This rise takes place only if protein synthesis occurs simultaneously. The cell content of deGTP does not change under any of the conditions of thymine starvation that have been investigated.

Regulation of the deo operon in Escherichia coli

SummaryThe synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.

Multiple regulation of nucleoside catabolizing enzymes: Regulation of thedeo operon by thecytR anddeoR gene products

SummaryThe protein and repressor nature of two regulatory gene products inE. coli has been demonstrated, employing mutants with either amber or thermosensitive mutations. The regulatory genes are thecytR and thedeoR genes, both of which contribute to the regulation of the synthesis of nucleoside catabolizing enzymes.Enzyme levels in strains with concurrent mutations in both regulatory genes are considerably higher than the sum of the levels in strains with acytR or adeo R mutation alone, indicating a certain co-operativity between the two repressor proteins.The glucose repression of enzyme levels observed in the double regulatory mutant is similar to that found in acytR mutant, and much more pronounced than the glucose effect in adeo R mutant.A model of the promoter-operator region in thedeo operon is proposed.

STUDIES ON THE ACID-SOLUBLE NUCLEOTIDE POOL IN THYMINE-REQUIRING MUTANTS OF ESCHERICHIA COLI DURING THYMINE STARVATION. I. ACCUMULATION OF DEOXYADENOSINE TRIPHOSPHATE IN ESCHERICHIA COLI 15 T-A-U-.

Abstract 1. 1. The variations in nucleotide content of Escherichia coli 15 T − A − U − (requiring thymine, arginine and uracil) during thymine starvation have been investigated. 90 min after removal of thymine from the medium a 2-fold increase in nucleotide content per cell mass was observed in HClO 4 extracts of culture. 2. 2. The time course of this increase, measured as absorbancy at 260 mμ, was investigated and was shown to be paralleled by a corresponding increase in purine-bound ribose, while purine-bound deoxyribose increased by the same factor, but at a much faster rate. 3. 3. Fractionation of the nucleotides on a column of Dowex-1 (Cl − form) showed that the increase in purine-bound deoxyribose was mainly due to an accumulation of deoxyadenosinetriphosphate and deoxyadenosinediphosphate. No similar increase in deoxyguanosine nucleotides was observed.

Thymidine breakdown and thymine uptake in different mutants of Escherichia coli

Abstract Incorporation experiments with [ 14 C]thymine into cells of an Escherichia coli strain indicate that thymine enters the metabolism of the cells through a trans - N -deoxyribosylase reaction rather than via thymidine phosphorylase. The cellular levels of these two enzymes have been estimated in cells and cell extracts of different mutants of Escherichia coli under varied growth conditions. The levels of the two enzymes are shown to be higher in the thymine-requiring mutants than in the wild-type parent strains. Both enzymes are released in the medium, when the cells are subjected to osmotic shock, indicating that these enzymes in the cell are located at or near the cell surface.

Mutants of Escherichia coli unable to metabolize cytidine: Isolation and characterization

SummaryStrains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.

Multiple regulation of nucleoside catabolizing enzymes: Effects of a polar dra mutation on the deo enzymes

SummaryStrains with an amber, polar mutation in the dra1 gene have been isolated. The mutation was introduced into a set of isogenic strains, wild type or with concurrent regulatory mutations, and further characterized by suppression and heat inactivation experiments.The effect of the polar dra mutation on the three remaining genes of the deo operon, the tpp, drm and pup genes, was determined by estimating the enzyme levels in the various dra-mutants. The effect was found to be non-coordinate, indicating the formation in the cells of two types of transcripts: A tetracistronic unit, containing the message from all four genes, and a dicistronic unit, covering the two distal genes only.

A deoxyuridine monophosphate phosphatase detected in mutants of Escherichia coli lacking alkaline phosphatase and 5′-nucleotidase

SummaryA two-step mutant lacking two periplasmic enzymes, alkaline phosphatase and 5′-nucleotidase, was prepared. In extracts of this strain phosphatase activities towards several 5′-nucleotides could be detected. We have partially purified a dUMP phosphatase by streptomycin precipitation and DEAE-chromatography. This preparation has significant phosphatase activity towards three substrates, viz. dUMP, dTMP and UMP, but only traces of activity towards nine other tested nucleoside monophosphates. The three activities are probably due to a single enzyme, since they decrease in parallel on long time storage at 4°C and respond in parallel to stimulatory and inhibitory influences of different buffers and metal ions. The best buffer tested is glycylglycine buffer; Mg2+ is required but 25% activity can be obtained with Co2+ and Mn2+ and 6–8% activity with Fe2+ and Ni2+. Ca2+, Cu2+ or Zn2+ inhibit the phosphatase activities in the presence of Mg2+ or Co2+. The activities towards UMP and dUMP in the DEAE—chromatography eluted identically. The enzyme has the remarkably high apparent Km of 10-2 M with all three substrates.

Cloning and Expression of Genes Encoding the nupG Nucleoside Transport System in Escherichia Coli

A DNA fragment, directing the synthesis of the nupG nucleoside transport system, has been cloned on a plasmid vector as evidenced by the complementation of chromosomal nupG mutations. The plasmid encoded transport system responded to the cytR and deoR control systems, which are known to act on the nupG transport system. Plasmid-containing minicells were used for specific in vitro synthesis of plasmid encoded proteins. Two proteins, with molecular mass of approximately 10,000 and 80,000, were detected, the occurrence and regulation of which suggested that they are components of the nupG nucleoside transport system.