Null thioredoxin and glutaredoxin Escherichia coli K-12 mutants have no enhanced sensitivity to mutagens due to a new GSH-dependent hydrogen donor and high increases in ribonucleotide reductase activity.
This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase. To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance. Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin. A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells. Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold. The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.
Miranda-Vizuete
A
A
Departamento de Genética, Universidad de Córdoba, Spain.
Martinez-Galisteo
E
E
Aslund
F
F
Lopez-Barea
J
J
Pueyo
C
C
Holmgren
A
A
D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005838:Genotype; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D016296:Mutagenesis; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D010088:Oxidoreductases; D011506:Proteins / Q000235:genetics*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013045:Species Specificity; D013879:Thioredoxins / Q000235:genetics*
D001426:Bacterial Proteins; D054477:Glutaredoxins; D009153:Mutagens; D011506:Proteins; D013879:Thioredoxins; D010088:Oxidoreductases; D012264:Ribonucleotide Reductases; D005978:Glutathione