Abstract
The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.
Highlights
The Levels of Trx, Grx1, and GSH Are Compensated—The levels of Trx and Grx1 were determined by specific competitive enzyme-linked immunoassays in E. coli strains defective in thioredoxin, glutaredoxin 1, and/or GSH biosynthesis (Table II, upper part)
Trx concentration seemed to have a higher effect on the Grx1 level than Grx1 on Trx, as expected from the existence of two other glutaredoxins, glutaredoxin 2 (Grx2) and glutaredoxin 3 (Grx3) (Åslund et al, 1994)
The putative involvement of lipoic acid in glutaredoxin reduction in vivo was investigated because dihydrolipoamide is a good reductant of E. coli Trx in vitro (Holmgren, 1979b)
Summary
Strain UC647 shows remarkable physiological properties when compared with its parental wild type cells It (i) grows on minimal medium plates; (ii) shows normal mutagenic sensitivity toward a wide variety of DNA-damaging agents; (iii) displays 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous RRase, respectively; and (iv) has very high levels (Ն20-fold increase) of ribonucleotide reductase activity. These properties indicate (i) that thioredoxin and glutaredoxin might not be essential for sulfate reduction, in contrast to previous data; (ii) that a balanced dNTP synthesis is maintained in the absence of thioredoxin and glutaredoxin, even under exposure to mutagens; and (iii) the existence of a third GSH-dependent hydrogen donor system for ribonucleotide reductase in E. coli. The grx::kan mutation, originally isolated by Russel and Holmgren (1988), will be referred in the text as grxA to indicate the absence of the classic glutaredoxin (Grx1), because two new additional glutaredoxins (Grx and Grx3) have been recently discovered (Åslund et al, 1994)
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