Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3

Abstract

Nitrite reductase catalyzes the reduction of nitrite to nitric oxide, the first step in denitrification to produce a gaseous product. We have cloned the gene nirK, which encodes the copper-type nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides, strain 2.4.3. The deduced open reading frame has significant identity with other copper-type nitrite reductases. Analysis of the promoter region shows that transcription initiates 31 bases upstream of the translation start codon. The transcription initiation site is 43.5 bases downstream of a putative binding site for a transcriptional activator. Maximal expression of a nirK-lacZ construct in 2.4.3 requires both a low level of oxygen and the presence of a nitrogen oxide. nirK-lacZ expression was severely impaired in a nitrite reductase-deficient strain of 2.4.3. This suggests that nirK expression is dependent on nitrite reduction. The inability of microaerobically grown nitrite reductase-deficient cells to induce nirK-lacZ expression above basal levels in medium unamended with nitrate demonstrates that changes in oxygen concentrations are not sufficient to modulate nirK expression.