Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli

Abstract

Summary: The gene (nir) encoding the copper-containing nitrite reductase (NIR) of a denitrifying bacterium, Alcaligenes faecalis S-6, was cloned by a synthetic oligonucleotide-probing method. The nucleotide sequence of the cloned DNA fragment revealed the primary structure of the NIR precursor containing the N-terminal signal sequence for secretion. A nucleotide sequence, possibly recognized by a transcriptional regulator resembling FNR was found upstream of the structural gene. When the cloned gene was expressed in Escherichia coli under the control of the lac promoter at 37 °C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20 °C, most of the NIR was detected in the soluble fraction and a significant portion of the protein was translocated into the periplasmic space, accompanied by removal of its signal sequence.