Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions

Abstract

By using a promoter probe plasmid we investigated expression of the linked nrdA and nrdB genes coding for the two different subunits of the ribonucleoside diphosphate reductase enzyme of Escherichia coli. For this reason, nrdA-lacZ, nrdAB-lacZ and nrdB-lacZ fusions were constructed. Results obtained indicate that the nrdB gene has a promoter from which it may be transcribed independently of the nrdA gene. Furthermore, the nrdB gene may also be transcribed from the nrdA promoter. The expression of the nrdB gene is about 14-fold higher from the nrdA promoter than from its own promoter. The induction of both nrdA and nrdB genes by DNA-damaging agents in the wild-type strain as well as in several SOS mutants was also studied; nrdA gene expression was increased by these treatments in RecA+, RecA-, and LexAInd- strains, although in both RecA- and LexAInd- mutants the nrdA gene expression was considerably lower than that in RecA+ cells. nrdB gene expression was stimulated by DNA damage only when its transcription was from the nrdA promoter, but there was no effect when nrdB was transcribed from its own promoter. In addition, the basal level of nrdA-lacZ and nrdAB-lacZ fusions was reduced in strains containing either RecA- and LexAInd- mutations or a multicopy plasmid carrying the lexA+ gene, whereas the presence of a LexA51Def mutation increased the constitutive expression of both fusions. On the contrary, the basal level of the nrdB-lacZ fusion remained constant in all these strains. Together these results indicate that induction of the SOS response enhances expression of the nrd genes from the nrdA promoter.