Open AccessConstruction and characterization of glutaredoxin-negative mutants of Escherichia coli.
Author(s) -
Marjorie Russel,
Arne Holmgren
Publication year - 1988
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.85.4.990
Subject(s) - glutaredoxin , ribonucleotide reductase , thioredoxin , biology , thioredoxin reductase , microbiology and biotechnology , escherichia coli , mutant , ribonucleotide , biochemistry , gene , nucleotide , protein subunit
Deoxyribonucleotides, the precursors of DNA, are formed de novo by ribonucleotide reductase, and in vitro thioredoxin or glutathione plus glutaredoxin have been isolated as hydrogen donors. The in vivo hydrogen donor for ribonucleotide reductase is not known. To study this, the Escherichia coli glutaredoxin gene (255 base pairs) was inactivated by inserting a 2-kilobase kanamycin-resistance fragment into the coding sequence of the cloned gene. The inactivated gene was inserted into the E. coli chromosome and mapped to about 18.5 min. A gene replacement technique was used to obtain a strain, A407, that lacked glutaredoxin by radioimmunoassay and by enzymatic assay with ribonucleotide reductase. Glutaredoxin was found not to be essential for viability of E. coli. Thioredoxin is also not essential for viability, as had been shown earlier, but a double mutant lacking glutaredoxin and thioredoxin could not be obtained by P1 transduction on a defined medium, indicating that either thioredoxin or glutaredoxin is essential. In rich medium, very slowly growing, unstable transductants were obtained that at high frequency gave rise to better growing cells. One such isolate, A410, was shown to still lack glutaredoxin and thioredoxin.