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.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...

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

“…norB-lacZ expression was only about 4% of the wild-type level in both strains. These levels are similar to those measured in a nirK mutant (34). It can also be seen that norB, norQ, and norD mutants exhibit reduced levels of nirK-lacZ expression in amended medium (Table 2).…”

“…Insertional inactivation of norB, norQ, and norD conferred the ability to induce high-level nirK-lacZ expression in unamended medium. In contrast, inactivation of nnrU or nnrT reduced the expression of nirK-lacZ in unamended medium to near-background levels, as also observed for nirK mutants (34).…”

“…The promoter sequence upstream of norC has been found in front of nirK and nnrR (34,35). Inactivation of nnrR affects expression of all four of these genes, suggesting that NnrR is the primary regulator of this set of genes.…”

“…Cells removed from stoppered flasks were not kept under anaerobic conditions but were used immediately for assays. Assays for Nir activity and the presence of NO 2 Ϫ in the culture medium were carried out as described by Tosques et al (34). To assay ␤-galactosidase and Nir activities, samples were taken at various times during the growth cycle.…”

“…NnrR, which is a member of the Fnr/Crp family of transcriptional regulators, regulates both Nor and NO 2 Ϫ reductase (Nir) expression but does not appear to be involved in regulation of other aspects of denitrification. In R. sphaeroides 2.4.3, expression of Nir and Nor is dependent on Nir activity, suggesting that NO production is critical for expression of proteins required for NO metabolism (34). Recent work has provided additional evidence that NO is an effector for expression of nirK, the Nir structural gene, and nor (20).…”