Abstract
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.
Highlights
This workinvestigates whether a mutator phenotype Escherichiacoli enzyme that reduces ribonucleotides to deis associated to the simultaneous deficiency in thiore- oxyribonucleotides required for DNA synthesis (Holmgren, doxin and glutaredoxin, the two known hydrogen do- 1985; Thelander and Reichard,1979; Reichard, 1988)
Thiorethe presence of thioredoxin and glutaredoxin may not doxin and glutaredoxin araelso important as dithiol reductants be absolutelyessential for sulfate reduction; (ii) showed in other reductive pathways, such asretdhuection of sulfate to normal mutagenicsensitivities toward a wide variety of sulfite via 3’-phosphoadenosine 5’-phosphosulfate (PAPS) re
GSH-dependent and 30%ofNADPH-dependent ribo- Absence of glutaredoxin does not produce any obvious growth nucleotide reduction capacity with CDP as substrate in defect in E. coli, and thioredoxin deficiency is associated with the presence or the absenceof exogenous ribonucleotide slow growth(Russeland Holmgren, 1988; Holmgren et al, reductase, respectively; and (iv)showed very high levels1978).Attempts to isolataestrain lackingboth thioredoxinand of ribonucleotide reductase activity,whichwas increased from 19- to 23-fold
Summary
All bacteria were E. coli K-1S2t.rain RK4349 (pro-3A (lac)6entA403 supE44h'his-218 rpsLlO9xyl-5 or xyl-7 ilvC7 metBl met E163::TnlO)was obtained from B. Strains A307 (K38 At&) and A407 (K38 grx::kan zbi::TnlO) were from A. Strain UC596 was constructed by PI (RK4349) x A307 transduction andselectionforTet'Met- (met E163::TnlO). Phagemediated transductions were carried out as described by Curtis (1981)
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