Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3.

A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.

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

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.

Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3.

During denitrification, the production and consumption of nitric oxide (NO), an obligatory and freely diffusible intermediate, must be tightly regulated in order to prevent accumulation of this highly reactive nitrogen oxide. Sequencing upstream of norCB, the structural genes for NO reductase, in the denitrifying bacterium Rhodobacter sphaeroides 2.4.3, we have identified a gene, designated nnrR, which encodes a protein that is a member of the cyclic AMP receptor family of transcriptional regulators. Insertional inactivation of nnrR prevents growth on nitrite, as well as the reduction of nitrite and NO, but has no effect on reduction of nitrate or photosynthetic growth. By using nirK-lacZ and norB-lacZ fusions, we have shown that NnrR is a positive transcriptional regulator of these genes. nnrR is expressed at a low constitutive level throughout the growth of R. sphaeroides 2.4.3. These results show that NnrR is not a global regulator but is instead a regulator of genes whose products are directly responsible for production and reduction of NO. Evidence is also presented suggesting that an NnrR homolog may be present in the nondenitrifying bacterium R. sphaeroides 2.4.1. The likely effector of NnrR activity, as determined on the basis of work detailed in this paper and other studies, is discussed.