Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

NosX function connects to nitrous oxide (N2O) reduction by affecting the Cu(Z) center of NosZ and its activity in vivo

The effect of loss of the 34-kDa periplasmic NosX protein on the properties of N2O reductase was investigated with an N2O-respiration negative, double mutant of the paralogous genes nosX and nirX of Paracoccus denitrificans. In spite of absence of whole-cell N2O-reducing activity, the purified reductase was catalytically active, which attributes NosX a physiological role in sustaining the reaction cycle. N2O reductase exhibited the spectroscopic features of Cu(A) and the redox-inert, paramagnetic state, Cu(Z)*, of the catalytic center. Cu(Z)*, hitherto considered the result of spontaneous reaction of the reductase with dioxygen, attains cellular significance.     

A NosA-specific bacteriophage can be used to select denitrification-defective mutants of Pseudomonas stutzeri

phi PS5, a double-stranded DNA bacteriophage of Pseudomonas stutzeri JM604 that adsorbs specifically to the outer-membrane protein NosA, was isolated from stagnant irrigation ditch water. Mutant strains that do not produce NosA are resistant to phi PS5 and cannot grow anaerobically with N2O as the sole electron acceptor. phi PS5 did not adsorb to nosA mutants and adsorption to the wild-type strain was reduced when cells were grown with a high concentration of copper, a condition that represses the synthesis of NosA. The isolation of spontaneous phi PS5-resistant mutants yielded strains that were clearly defective in growth on N2O at about a 10% incidence. About half of these strains could respire N2O when supplied with a high concentration of exogenous copper.     

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.     

Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration

Bacterial nitrous oxide (N2O) respiration depends on the polytopic membrane protein NosR for the expression of N2O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX3CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N2O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N2O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N2O respiration. We also discuss its putative functions in electron donation and redox activation.     

Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities.     

Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri

By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N(2)O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.     

Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities.     

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 μmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 μmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.     

Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum

The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).     

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.     

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.     

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.