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.

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.The availability of fixed nitrogen is often a major factor controlling the biological productivity of ecosystems. During the cycling of nitrogen in the biosphere, a significant sink for fixed nitrogen is denitrification, the reduction of nitrate to gaseous forms of nitrogen, primarily nitrogen gas (22). Gaseous forms of nitrogen are unavailable for use by the majority of organisms. Because denitrification is a process that results in the conversion of fixed forms of nitrogen to gaseous forms, it can have a significant impact on the productivity of an ecosystem. For example, nitrate concentrations in the ocean have been found to be a major factor limiting biological productivity (19). Denitrification is the major sink for ocean nitrate, establishing a direct link between denitrification and biological productivity in the marine environment (6). This has been dramatically demonstrated in recent studies that have shown that decreases in the rate of denitrification during the last glacial maximum may have increased the productivity of the ocean enough to lower the partial pressure of carbon dioxide in the atmosphere (1, 9).Denitrification is a respiratory process in which bacteria utilize nitrate and other inorganic oxides of nitrogen as alternate electron acceptors when oxygen concentrations are limiting. In the first step of denitrification, nitrate is reduced to nitrite (12). This reaction is not unique to denitrification, however, since it occurs during ammonification and assimilatory nitrate reduction. The next step in denitrification is the reduction of nitrite to nitric oxide (NO). This reaction is catalyzed by nitrite reductase and is the defining reaction of denitrification, since it produces the first gaseous intermediate (38). Moreover, nitric oxide-producing nitrite reductases are associated only with denitrification (12). There are two classes of nitrite reductase...

Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with prrA restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK. Inactivation of ccoN, part of the cbb 3 -type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity. This was unexpected, since PrrA-P accumulates in the ccoN strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK.

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.Denitrification is the reduction of nitrate (NO 3 Ϫ ) to gaseous intermediates, principally nitrogen gas. Nitric oxide (NO), an obligatory intermediate during denitrification, is generated from the one-electron reduction of nitrite (NO 2 Ϫ ) (41). NO reduction is coupled to energy generation (18,29). NO is also a well-known cytotoxic compound, so its production during denitrification has the potential of causing significant cell damage. To mitigate the toxicity of NO, its steady-state concentration during denitrification is maintained at low-nanomolar levels (11). The protein responsible for NO reduction, NO reductase (Nor), catalyzes the reaction 2NO ϩ 2H ϩ 3N 2 O ϩ H 2 O. Nitrous oxide (N 2 O) is an inert, nontoxic intermediate that is frequently the terminal product of denitrification (42). Nor has been purified and shown to be a heterodimeric membrane protein (9,14,16). Metal analysis has shown that it contains only iron in stoichiometric amounts. Recently, the Nor structural genes have been characterized, and sequence analysis revealed that Nor was related to the cytochrome c oxidase superfamily (36). In particular, Nor is most closely related to the heme b-containing oxidases, which are expressed under conditions of low oxygen concentration. It has been suggested that Nor was the original member of this family and that the other members arose by modifying the Nor structure (26).The genetic organization of the region of the chromosome encoding the nor structural genes varies among denitrifiers. In Pseudomonas stutzeri, the two structural genes form a distinct transcriptional unit (43). In Pseudomonas aeruginosa, the two structural genes ...