Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin

Abstract

Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized. The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase. In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase. Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele. The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al. (J.-O. Hoog, H. von Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43:13-21, 1986).

The Role of the Thioredoxin and Glutaredoxin Pathways in Reducing Protein Disulfide Bonds in the Escherichia coliCytoplasm

“…None of these strains required cysteine or glutathione to grow in NZ-amine-A medium. Similar findings have been reported for trxB grxA and trxA grxA mutants and were attributed to the inability of these strains to reduce PAPS reductase, an enzyme that is required for growth when sulfate is the only source of sulfur in the medium (32,38,39).…”

“…Previous reports suggested that trxB gor and trxB gshA strains do not exhibit an aerobic growth defect, although the trxB gor strain described was not viable at 42°C (38,49). Suppressor mutations may have been unwittingly selected during the manipulations of these strains.…”

Glutaredoxin homolog encoded by vaccinia virus is a virion-associated enzyme with thioltransferase and dehydroascorbate reductase activities.

“…Genetic and biochemical studies suggest that some GRXs are cofactors for ribonucleotide reductase (5,(31)(32)(33). Indeed, the encoding of both subunits of ribonucleotide reductase by vaccinia virus provides a rationale for the presence of a viral grx.…”

Molecular and Cellular Aspects of Thiol–Disulfide Exchange

“…However, oxidized glutaredoxin is reduced directly by cellular glutathione, and the resulting GSSG is, in turn, reduced by NADPH via the glutathione reductase reaction (69). Recent experiments have suggested that glutaredoxin may support most of the ribonucleotide reduction under normal conditions in E. cofi (97).…”