Jan Neuhard

Studies on the acid-soluble nucleotide pool in thymine-requiring mutants of Escherichia coli during thymine starvation: III. On the regulation of the deoxyadenosine triphosphate and deoxycytidine triphosphate pools of Escherichia coli

Abstract Changes in the size of the deoxyribonucleoside triphosphate pool have been investigated in mutants of Escherichia coli subjected to different conditions of thymine restriction. The following results were obtained. 1. 1. Five different thymine-requiring mutants of E. coli , after removal of thymine from their growth media, all showed an immediate rise in dATP content reaching a maximum after about 60 min. 2. 2. In two mutants (both E. coli 15 T- mutants) the dCTP pool increased linearly 10- to 15-fold after a lag of about 30 min. In these two mutants the maximal amount of dATP accumulated was only half of that reached by the other three. 3. 3. Thymine prototrophs of E. coli also accumulated dATP when deprived of their intracellular thymidine nucleotides by addition of 5-fluorodeoxyuridine. 4. 4. Inhibition of DNA synthesis in E. coli 15 T-A-U- by novobiocin in the presence of thymine did not result in any significant changes in the deoxyribonucleoside triphosphate pools. The results are discussed in relation to the regulation of deoxyribonucleotide synthesis.

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.

Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.

Studies on the acid-soluble nucleotide pool in Escherichia coli: IV. Effects of hydroxyurea

Abstract Addition of hydroxyurea to exponentially-growing cultures of Escherichia coli resulted in a concentration-dependent decrease in the acid-soluble deoxyribonucleoside triphosphate pools of the cells. At the same time a corresponding relative decrease in the rate of DNA synthesis was observed, suggesting that the limiting factor for DNA synthesis in hydroxyurea-treated cultures of E. coli is the size of the precursor pool. In contrast, the ribonucleoside triphosphate pools did not change significantly during treatment with the drug.

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

SummaryThe Bacillus subtilis cdd gene encoding cytidine/2′-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in λD69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis δ43 and δ37 RNA polymerase holoenzymes during growth and stationary phase.

RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC.

A mutant of Salmonella typhimurium with a defect in the regulation of pyr‐gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5‐fluorouracil and 5‐fluorouridine. The mutant possesses 4‐fold elevated pools of the pyrimidine nucleoside triphosphatases, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40‐fold and 7‐fold higher in the mutant than in the parent strain when grown in minimal media. Furthermore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5′‐monophosphate decarboxylase are approximately 3‐fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the beta and beta’ subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control of pyr‐gene expression in S. typhimurium.

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.

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase.

Rifampicin‐resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil‐sensitive). The uracil‐sensitive phenotype (UraS) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH, which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta‐subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.

Steroid inhibition of reduced diphosphopyridine nucleotide-oxidase activity in electron-transport particles I. Kinetic studies

Abstract When deoxycorticosterone and several other steroid hormones are added to a suspension of electron-transport particles oxidizing DPNH, the rate of oxidation spontaneously decreases. The influence of experimental conditions on this inhibitory effect has been studied. It was found that the inhibition is independent of the level of specific activity of the oxidase and of prolonged storage at −15° of the electron-transport particles preparation. The composition of the reaction medium, however, especially with respect to buffer concentration, influences the degree of inhibition. The interaction of steroid and enzyme is reversible and the inhibition is non-competitive. The concentrations at which seven corticosteroids produce 50% inhibition have been determined. The results together with results of experiments, in which the distribution of four corticosteroids between electron-transport particles and suspension medium was estimated, indicated that the differences among the steroids with respect to their potency as inhibitors of the DPNH-oxidase activity of electron-transport particles reflect differences in their solubility in electron-transport particles more than differences in the affinity of the enzyme per se for the individual steroid.

Deciphering the function of an ORF: Salmonella enterica DeoM protein is a new mutarotase specific for deoxyribose

We identified in Salmonella enterica serovar Typhi a cluster of four genes encoding a deoxyribokinase (DeoK), a putative permease (DeoP), a repressor (DeoQ), and an open reading frame encoding a 337 amino acid residues protein of unknown function. We show that the latter protein, called DeoM, is a hexamer whose synthesis is increased by a factor over 5 after induction with deoxyribose. The CD spectrum of the purified recombinant protein indicated a dominant contribution of βtype secondary structure and a small content of α‐helix. Temperature and guanidinium hydrochloride induced denaturation of DeoM indicated that the hexamer dissociation and monomer unfolding are coupled processes. DeoM exhibits 12.5% and 15% sequence identity with galactose mutarotase from Lactococcus lactis and respectively Escherichia coli, which suggested that these three proteins share similar functions. Polarimetric experiments demonstrated that DeoM is a mutarotase with high specificity for deoxyribose. Site‐directed mutagenesis of His183 in DeoM, corresponding to a catalytically active residue in GalM, yielded an almost inactive deoxyribose mutarotase. DeoM was crystallized and diffraction data collected for two crystal systems, confirmed its hexameric state. The possible role of the protein and of the entire gene cluster is discussed in connection with the energy metabolism of S. enterica under particular growth conditions.