Transcriptional regulation of nrdAB genes in E. coli – analysis of the new regulator NrdR

Abstract

The enzyme ribonucleotide reductase (RNR) provides the building blocks necessary for DNA synthesis and repair. In E. coli there are three classes of RNRs. The oxygen dependent Ia reductase is encoded by the nrdAB gene, and its transcriptional regulation by NrdR is the target of this study. We experimentally demonstrated negative regulation of nrdAB expression by NrdR, using plasmids with nrdAB promoter gfp fusion in wild type and mutant (ΔnrdR) strains. This repressor is a link between RNR inhibition with hydroxyurea and increased expression of nrdAB in E. coli. We show that the expression of nrdAB promoter reveal a 3-fold lower nrdAB promoter activity in wild type than in mutant which shows the effect of NrdR repression. An increase by 2.4 fold in wt strain with plasmid containing all promoter elements (p34) treated with HU and 1.1 fold in mutant was observed. In addition, the presence of the intact plasmid (p38) in wt strain show 1.4 fold increase with addition of HU whereas in mutant only 1.1. The tight regulation of RNR and dNTP pools is monitored by Flow cytometry analysis where DNA/cell mass decrease was observed in wt and mutant strains with HU. The Flow cytometry also showed that DNA replication occurs faster in mutant strains than in the wild type. Finally we show that inhibition of DNA-synthesis, by way of treatment with the chemical nalidixic acid, greatly stimulates expression of nrdAB, and strengthens the repressive efficacy of the NrdR. 3