The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr ؉ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.

Denitrification has been known for more than a century and is widely recognized as a key process in the biogeochemical nitrogen cycle. It is the major mechanism that converts combined nitrogen, the form available to eukaryotes, to dinitrogen gas, thereby completing the nitrogen cycle. In recent years, denitrification has taken on added importance for the following reasons. First, it is a major source of NO and N20, gases that are of focal importance to atmospheric ozone destruction and to global warming. Indeed, N20 concentrations in the atmosphere have been increasing at 0.2 to 0.3% per year for at least 20 to 30 years (44), and N20 along with CO2 and CH4 are the most important gases thought to be driving climate change. More recently, significant fluxes of NO from soils to the atmosphere have been measured (13), raising questions about the microbial sources of this gas. Second, denitrification is important to waste treatment as a means of both removing excess nitrate and stimulating carbon removal when aeration is difficult. In the latter case, there is increased interest in using nitrate to drive pollutant bioremediation in aquifers (33), because nitrate is more water soluble and mobile in soil than is oxygen. Third, denitrification below the rooting zone has been largely ignored, but recent evidence shows that it is important to an understanding of the carbon, nitrogen, and mineral cycling in the vadose zone, aquifers, and deeper geological formations. Fourth, the discovery that NO is a key chemical signal in a variety of mammalian functions, including the cell killing function of macrophages, neurotransmission, and control of smooth muscle, led to NO being named 1992 Molecule of the Year by Science magazine (37). Whether microbial colonizers of mammals play any role in production or consumption of bioactive NO is unknown. Similarly, whether there is any mechanistic insight to be gained by comparing microbial and mammalian NO binding or transformation is just beginning to be explored. These reasons all speak to the importance of gaining a basic understanding of denitrification. This review summarizes recent advances in the physiology, biochemistry, and genetics of the central steps in denitrification, nitrite and nitric oxide reduction. NITRIC OXIDE AS AN INTERMEDIATE IN DENITRIFICATION

Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had TnS inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir-mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with '5NO in H2180, about 25% of the oxygen found in nitrous oxide exchanged with H20. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 180 from H218O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the '8O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.Dissimilatory reduction of nitrite is the key step in the denitrification pathway; it is the point of divergence from assimilatory nitrogen metabolism (24). There are two types of nitrite reductases: one contains the cytochromes cdl, and the other contains copper (6,14,20). Organisms containing the cytochrome cd, nitrite reductases are more frequently isolated from nature, whereas Cu-type nitrite reductases are found in organisms that exhibit more phylogenic diversity and occupy a wider range of ecological niches (6, 9).Nitric oxide is the major product of nitrite reduction by purified nitrite reductases, and nitrous oxide is a minor product (4,14,17,26). NO is generally accepted as one of the free and obligatory intermediates in the denitrification pathway (2,5, 8,10,14,20,28), and NO reductases have been purified from Pseudomonas stutzeri Zobell (13) and Paracoccus denitnificans (4, 7). We recently showed that many denitrifiers containing Cu or cdl nitrite reductases are capable of undergoing 0-atom exchange with H2180 during the reduction of NO to N20 (27) and that a labeled intermediate can be trapped with azide. These results suggest that a nitrosyl complex is formed during the reaction. It was also shown that the extent of the 0-atom exchange reaction depended on the availability of electrons. The dependence of nitrosyl transfer upon the presence of NO during reduction of N02-has been demonstrated (11).TnS was used by Zumft et al. (29) to generate mutants deficient in dissimilatory nitrite reduction (Nir-) in P. stutzeri Zobell. All of the mutants isolated possessed normal NO reduction activity, indicating that NO reduction and nitrite reduction are distinct. The nitrite reductase gene (nirS) from Pseudomonas aeruginosa (21) and from two strains of P. stutzeri (Zobell [15]

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper-and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putativeJfr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductases containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d, showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.

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