Molecular cloning and DNA sequence of dniR, a gene affecting anaerobic expression of the Escherichia coli hexaheme nitrite reductase.

Abstract

A gene responsible for increased synthesis of hexaheme nitrite reductase (cytochrome c552) of Escherichia coli K-12 was cloned into pBR322 by the direct immunological screening method using antiserum against the purified enzyme. The cloned gene was mapped at 5 min on the chromosomal linkage map as the dni gene (related to increased synthesis of the dissimilatory nitrite reductase) by conjugation and transduction. The dni gene was subcloned into pUC118 and was shown to be on a 2.6-kilobase-pair PstI-BamHI fragment by immunoblotting analysis of the expressed enzyme. The nucleotide sequence of this fragment was determined. A plausible open-reading frame corresponding to 222 amino acids was detected. Analysis of a dni deletion mutant by immunoblotting demonstrated that this mutant expressed a greatly reduced amount of the nitrite reductase. Thus, the dni gene is suggested to have a positive regulatory action on induced synthesis of the nitrite reductase, and was designated as dniR.