CLONING AND CHARACTERIZATION OF A NITRITE REDUCTASE GENE FROM ALCALIGENES-FAECALIS AND ITS EXPRESSION IN ESCHERICHIA-COLI

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-degrees-C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20-degrees-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.