Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.

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 putative fnr 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 reductase containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d1 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.     

A spectrophotometric assay for dissimilatory nitrite reductases

A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent. The copper-containing nitrite reductase from Achromobacter cycloclastes has been shown to have apparent Km's for reduced cytochrome c and nitrite of 86 +/- 5 and 5.63 +/- 0.03 microM, respectively. The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent Km's for reduced cytochrome c and nitrite of 260 +/- 60 and 1.8 +/- 0.8 microM, respectively. This assay represents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes.     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo‐containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane‐bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.     

Structural gene (nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16.

Denitrification by Alcaligenes eutrophus H16 is genetically linked to megaplasmid pHG1. Unexpectedly, the gene encoding the nitrite reductase (nirS) was identified on chromosomal DNA. The nirS product showed extensive homology with periplasmic nitrite reductases of the heme cd1-type. Disruption of nirS abolished nitrite-reducing ability, indicating that NirS is the enzyme essential for denitrification in A.eutrophus.     

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.     

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.     

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N₂O reductase and nitrite reductase (cytochrome cd ₁) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N₂O reductase in a mutant strain deficient in the chromophore of N₂O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.     

Detection,with a pH indicator,of bacterial mutants unable to denitrify

In order to facilitate isolation of mutants with alterations in the denitrification pathway, a new screening procedure using phenol red incorporated into agar overlay has been defined. Alkalinization in the neighbourhood of denitrifying colonies respiring nitrate or nitrite gives rise to a red circular halo. Antimycin blocked these colour changes, which suggests their association with the periplasmic reduction of nitrite. Inhibition of nitrous oxide reductase by acetylene had no significant effect on alkalinization elicited by nitrate or nitrite. Several mutants negative by the phenol red staining test were generated by transposon Tn5 mutagenesis of Paracoccus denitrificans. All these mutants were defective in the activities of nitrite and nitric oxide reductases while the other denitrification activities were present at the wild-type level.     

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.     

A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Interrelations of micro-organisms and mulberry

Summary Evidence has been presented to show that the autotrophic nitrifying organisms get stimulated in the mulberry rhizosphere. Three species of Pseudomonas, one each of Achromobacter and Bacillus capable of degrading methionine were shown to be stimulated in the rhizosphere. These bacteria were capable of reversing the inhibitory effect of methionine on soil nitrification. Two of them were able to form nitrite from methionine. The possibility that the increased nitrifying activity in the mulberry rhizosphere in the presence of methionine found in mulberry root exudations was the result of the activity of these organisms was suggested.     

Specific sodium dependence of a nitrate reductase in a marine bacterium

Abstract The two key enzymes of denitrification, the nitrate and nitrate reductases, were studied in a marine organism, Pseudomonas nautica , strain 617. Both enzymes were grown under anaerobic conditions with either nitrate or nitrous oxide as electron acceptor. The effect of sodium on these enzymes was studied. Only sodium activated the membranous and purified nitrate reductase, and none of the salts affected nitrite reductase.     

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O₂ pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids. Received: 17 April 1996 / Accepted: 28 May 1996     

Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.