Copper-containing Nitrite Reductase Research Articles

Metagenome Analysis of a Complex Community Reveals the Metabolic Blueprint of Anammox Bacterium “Candidatus Jettenia asiatica”

Anaerobic ammonium-oxidizing (anammox) bacteria are key players in the global nitrogen cycle and responsible for significant global nitrogen loss. Moreover, the anammox process is widely implemented for nitrogen removal from wastewaters as a cost-effective and environment-friendly alternative to conventional nitrification-denitrification systems. Currently, five genera of anammox bacteria have been identified, together forming a deep-branching order in the Planctomycetes-Verrucomicrobium-Chlamydiae superphylum. Members of all genera have been detected in wastewater treatment plants and have been enriched in lab-scale bioreactors, but genome information is not yet available for all genera. Here we report the metagenomic analysis of a granular sludge anammox reactor dominated (∼50%) by “Candidatus Jettenia asiatica.” The metagenome was sequenced using both Illumina and 454 pyrosequencing. After de novo assembly 37,432 contigs with an average length of 571 nt were obtained. The contigs were then analyzed by BLASTx searches against the protein sequences of “Candidatus Kuenenia stuttgartiensis” and a set of 25 genes essential in anammox metabolism were detected. Additionally all reads were mapped to the genome of an anammox strain KSU-1 and de novo assembly was performed again using the reads that could be mapped on KSU-1. Using this approach, a gene encoding copper-containing nitrite reductase NirK was identified in the genome, instead of cytochrome cd1-type nitrite reductase (NirS, present in “Ca. Kuenenia stuttgartiensis” and “Ca. Scalindua profunda”). Finally, the community composition was investigated through MetaCluster analysis, 16S rRNA gene analysis and read mapping, which showed the presence of other important community members such as aerobic ammonia-oxidizing bacteria, methanogens, and the denitrifying methanotroph “Ca. Methylomirabilis oxyfera”, indicating a possible active methane and nitrogen cycle in the bioreactor under the prevailing operational conditions.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of GK0767, the copper-containing nitrite reductase from Geobacillus kaustophilus.

The soluble region (residues 32-354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426, has been cloned and overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.3 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5 Å. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V(M) value of 3.14 Å(3) Da(-1).

Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Abstract Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum–Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the 15N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.

The Possible Involvement of Copper-Containing Nitrite Reductase (NirK) and Flavohemoglobin in Denitrification by the FungusCylindrocarpon tonkinense

The occurrence of denitrification and nitrate respiration among eukaryotes has been established during the last few decades. However, denitrification-related eukaryotic genes have been isolated from only a few fungi, and eukaryotic denitrification (or nitrate respiration) is still inadequately understood. In this study, we identified genes that were up-regulated under denitrifying conditions in the fungus Cylindrocarpon tonkinense using the suppression subtraction hybridization technique, and the expression patterns of these genes were characterized by Northern analysis. We identified copper-containing nitrite reductase, cytochrome P450 nitric oxide reductase, flavohemoglobin (Fhb), and formate/nitrite transporter homolog genes as possibly involved in fungal denitrification. Our results concerning the involvement of Fhb and formate/nitrite transporter perhaps provide new insight into the fungal denitrification system.

Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense

In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.

A Eukaryotic Copper-Containing Nitrite Reductase Derived from a NirK Homolog Gene ofAspergillus oryzae

We cloned a bacterial copper-containing nitrite reductase (NirK) homolog gene of Aspergillus oryzae (AonirK). Alignment showed that amino acid residues crucial for copper binding are conserved in the deduced sequence of the fungal protein. The recombinant protein exhibited distinct nitrite reductase activity, and its absorption and EPR spectra showed the presence of type 1 and type 2 copper atoms in the molecule. AonirK transcriptionally responded to denitrification conditions. Although the denitrifying activity of A. oryzae was weak under the conditions employed, high expression of the gene in the fungal cells enhanced the denitrifying activity 6-fold, accompanied by distinct cell growth. Furthermore, the highly expressed AoNirK was subcellularly localized to the mitochondria. The results demonstrated that AoNirK is responsible for fungal denitrification. Discussion is added on the novel insight concerning the origin and evolution of the mitochondrion provided by the findings for eukaryotic NirKs.

Nitrous oxide emission from nitrifying activated sludge dependent on denitrification by ammonia-oxidizing bacteria

Nitrous oxide (N 2O) is emitted during the aerated nitrification process of wastewater treatment, but its mechanism is not understood. In this study, we employed a model system to clarify the mechanism of N 2O emission, utilizing the activated sludge derived from a piggery effluent. Aerated incubation of the sludge with ammonium ( NH 4 + ) or hydroxylamine (NH 2OH) resulted in the emission of a significant amount of N 2O. The emission stopped when the nitrification substrate ( NH 4 + or NH 2OH) was exhausted. When NH 4 + was replaced with nitrate ( NO 3 - ) and nitrite ( NO 2 - ), no N 2O was emitted. This result suggests that the N 2O emission under nitrifying conditions did not depend on the oxidation of NO 2 - by nitrite-oxidizing bacteria (NOB) or denitrification by heterotrophic denitrifiers but depended on the oxidation of NH 4 + by ammonia-oxidizing bacteria (AOB). When NO 2 - , the product of nitrification by AOB, was added to the NH 4 + -oxidizing system, N 2O emission was enormously enhanced, suggesting that N 2O was formed via denitrification. Diethyldithiocarbamate (DCD), an inhibitor of copper-containing nitrite reductase (NirK), strongly blocked N 2O emission from NH 2OH. Furthermore, the expression of the gene ( nirK) encoding NirK of AOB was detected in the sludge exposed to the nitrifying conditions. The results showed that N 2O emission during the nitrification process depends on denitrification by AOB that reside in the activated sludge. This study provides direct evidence for the cause of N 2O emission from activated sludge (non-pure culture).

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.

Structural basis of inter-protein electron transfer for nitrite reduction in denitrification

Recent earth science studies have pointed out that massive acceleration of the global nitrogen cycle by anthropogenic addition of bio-available nitrogen has led to a host of environmental problems. Nitrous oxide (N(2)O) is a greenhouse gas that is an intermediate during the biological process known as denitrification. Copper-containing nitrite reductase (CuNIR) is a key enzyme in the process; it produces a precursor for N(2)O by catalysing the one-electron reduction of nitrite (NO2-) to nitric oxide (NO). The reduction step is performed by an efficient electron-transfer reaction with a redox-partner protein. However, details of the mechanism during the electron-transfer reaction are still unknown. Here we show the high-resolution crystal structure of the electron-transfer complex for CuNIR with its cognate cytochrome c as the electron donor. The hydrophobic electron-transfer path is formed at the docking interface by desolvation owing to close contact between the two proteins. Structural analysis of the interface highlights an essential role for the loop region with a hydrophobic patch for protein-protein recognition; it also shows how interface construction allows the variation in atomic components to achieve diverse biological electron transfers.

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO2-) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 +/- 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H2O and D2O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of approximately 1.33 +/- 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H2O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mM. As a consequence, the observed ET process was faster in D2O than in H2O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of approximately 0.61 +/- 0.02 between 3 and 10 mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.

Cytochrome c551 Is a Mediator of Electron Transfer between Copper-Containing Nitrite Reductase and Azurin in a Denitrifying Bacterium, Achromobacter xylosoxidans

Abstract Cu-containing nitrite reductase from a denitrifying bacterium, Achromobacter xylosoxidans GIFU1051 (the organism formerly known as Alcaligenes xylosoxidans), can accept an electron from three possible electron donors (cytochrome c551 and two azurins). We have kinetically demonstrated that cytochrome c551 functions as a mediator in the electron flow processes between the enzyme and two azurins.

Electroreduction of nitrite to nitrogen oxide by a copper-containing nitrite reductase model complex incorporated into collagen film

The electrocatalytic reduction of nitrite to NO by CuMe 2bpaCl 2, which is a model for the active site of copper-containing nitrite reductase, incorporated into collagen film was investigated. The 77-K EPR spectrum of CuMe 2bpaCl 2 in the collagen matrix revealed the typical axial signals ( g // = 2.26, g ⊥ = 2.05, A // = 16.4 mT) of a tetragonal Cu 2+ chromophore. The redox potential, which is related to the Cu +/Cu 2+ couple, was − 63 mV ( ∆E = 72 mV) at pH 5.5. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of CuMe 2bpaCl 2 in the collagen matrix. Upon reaching − 300 mV, a linear generation of NO was observed for the CuMe 2bpaCl 2/collagen film-coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed using the Michaelis–Menten equation, where V max = 3.16 nM s − 1 and K m = 1.1 mM at pH 5.5. The current increase and the reaction rate were dependent on the pH of the solution. The mechanism of nitrite reduction by the copper complex in the collagen matrix was the same mechanism as that of the enzyme in aqueous solution.

Plant presence and species combination, but not diversity, influence denitrifier activity and the composition ofnirK-type denitrifier communities in grassland soil

To explore potential links between plant communities, soil denitrifiers and denitrifier function, the impact of presence, diversity (i.e. species richness) and plant combination on nirK-type denitrifier community composition and on denitrifier activity was studied in artificial grassland plant assemblages over two consecutive years. Mesocosms containing zero, four and eight species and different combinations of two species were set up. Differences in denitrifier community composition were analysed by canonical correspondence analyses following terminal restriction fragment length polymorphism analysis of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. As a measure of denitrifier function, denitrifier enzyme activity (DEA) was determined in the soil samples. The presence as well as the combination of plants and sampling time, but not plant diversity, affected the composition of the nirK-type denitrifier community and DEA. Denitrifier activity significantly increased in the presence of plants, especially when they were growing during summer and autumn. Overall, we found a strong and direct linkage of denitrifier community composition and functioning, but also that plants had additional effects on denitrifier function that could not be solely explained by their effects on nirK-type denitrifier community composition.

Eukaryotic nirK Genes Encoding Copper-Containing Nitrite Reductase: Originating from the Protomitochondrion?

Although denitrification or nitrate respiration has been found among a few eukaryotes, its phylogenetic relationship with the bacterial system remains unclear because orthologous genes involved in the bacterial denitrification system were not identified in these eukaryotes. In this study, we isolated a gene from the denitrifying fungus Fusarium oxysporum that is homologous to the bacterial nirK gene responsible for encoding copper-containing nitrite reductase (NirK). Characterization of the gene and its recombinant protein showed that the fungal nirK gene is the first eukaryotic ortholog of the bacterial counterpart involved in denitrification. Additionally, recent genome analyses have revealed the occurrence of nirK homologs in many fungi and protozoa, although the denitrifying activity of these eukaryotes has never been examined. These eukaryotic homolog genes, together with the fungal nirK gene of F. oxysporum, are grouped in the same branch of the phylogenetic tree as the nirK genes of bacteria, archaea, and eukaryotes, implying that eukaryotic nirK and its homologs evolved from a single ancestor (possibly the protomitochondrion). These results show that the fungal denitrifying system has the same origin as its bacterial counterpart.

Expression of a putative nitrite reductase and the reversible inhibition of nitrite‐dependent respiration by nitric oxide in Nitrobacter winogradskyi Nb‐255

The nitrite oxidizing Alphaproteobacterium, Nitrobacter winogradskyi, primarily conserves energy from the oxidation of nitrite (NO(2)(-))to nitrate (NO(3)(-)) through aerobic respiration. Almost 20 years ago, NO-dependent NADH formation was reported to occur in both aerobic and anaerobic cell suspensions of N. winogradskyi strain 'agilis', suggesting that NO oxidation might contribute to energy conservation by Nitrobacter. Recently, the N. winogradskyi Nb-255 genome was found to contain a gene (Nwin_2648) that encodes a putative copper-containing nitrite reductase (NirK), which may reduce NO(2)(-) to NO. In this study, the putative nirK was found to be maximally transcribed under low O(2) (between zero and 4% O(2)) in the presence of NO(2)(-). Transcription of nirK was not detected under anaerobic conditions in the absence of NO(2)(-) or in the presence of NO(3)(-) and pyruvate. Although net production of NO could not be detected from either aerobically grown or anaerobically incubated cells, exogenous NO was consumed by viable cells and concomitantly inhibited NO(2)(-)-dependent O(2) uptake in a reversible, concentration dependent manner. Both NO(2(-)-dependent O(2) uptake and NO consumption were inhibited by 1 mM cyanide suggesting involvement of cytochrome oxidase with NO consumption. Abiotic consumption of NO was measured, yet, both the rates and kinetics of NO transformation in buffer alone, or by heat killed, or cyanide-treated cells differed from those of viable cells. In light of this new information, a modified model is proposed to explain how NirK and NO manage electron flux in Nitrobacter.

Habitat partitioning of denitrifying bacterial communities carrying nirS or nirK genes in the stratified water column of Lake Kinneret, Israel

The community composition of denitrifying bacteria was studied in the stratified water column of Lake Kinneret. The nitrite reductase genes nirS and nirK were amplified by PCR from water samples taken at 1, 14, 19 and 22 m depth, which represent the epi-, meta- and hypolimnion of the lake. The PCR products were analyzed with terminal restriction fragment length polymorphism (T-RFLP) and clone libraries. The highest diversity of nirS denitrifying communities was observed at 1 m depth. According to the T-RFLP profiles and clone libraries of nirS products, 2 groups of denitrifiers were common to and dominant in all depths. Deduced protein sequences from one of these groups displayed low identity (77%) with other nirS sequences reported in GenBank. Denitrifying bacterial communities with nirK were most diverse at 22 m and showed highest similarity to those at 19 m depth. Sequences unrelated to nirK dominated the clone libraries from 1 m depth, suggesting that denitrifying bacteria with copper-containing nitrite reductase were less frequent at this depth. The results suggest that microorganisms with nirK and those with nirS respond differently to the environmental conditions in the stratified water column of Lake Kinneret.

Effect of a Tridentate Ligand on the Structure, Electronic Structure, and Reactivity of the Copper(I) Nitrite Complex: Role of the Conserved Three-Histidine Ligand Environment of the Type-2 Copper Site in Copper-Containing Nitrite Reductases

It is postulated that the copper(I) nitrite complex is a key reaction intermediate of copper containing nitrite reductases (Cu-NiRs), which catalyze the reduction of nitrite to nitric oxide (NO) gas in bacterial denitrification. To investigate the structure-function relationship of Cu-NiR, we prepared five new copper(I) nitrite complexes with sterically hindered tris(4-imidazolyl)carbinols [Et-TIC = tris(1-methyl-2-ethyl-4-imidazolyl)carbinol and iPr-TIC = tris(1-methyl-2-isopropyl-4-imidazolyl)carbinol] or tris(1-pyrazolyl)methanes [Me-TPM = tris(3,5-dimethyl-1-pyrazolyl)methane; Et-TPM = tris(3,5-diethyl-1-pyrazolyl)methane; and iPr-TPM = tris(3,5-diisopropyl-1-pyrazolyl)methane]. The X-ray crystal structures of all of these copper(I) nitrite complexes were mononuclear eta(1)-N-bound nitrite complexes with a distorted tetrahedral geometry. The electronic structures of the complexes were investigated by absorption, magnetic circular dichroism (MCD), NMR, and vibrational spectroscopy. All of these complexes are good functional models of Cu-NiR that form NO and copper(II) acetate complexes well from reactions with acetic acid under anaerobic conditions. A comparison of the reactivity of these complexes, including previously reported (iPr-TACN)Cu(NO2) [iPr-TACN = 1,4,7-triisopropyl-1,4,7-triazacyclononane], clearly shows the drastic effects of the tridentate ligand on Cu-NiR activity. The copper(I) nitrite complex with the Et-TIC ligand, which is similar to the highly conserved three-histidine ((His)3) ligand environment in the catalytic site of Cu-NiR, had the highest Cu-NiR activity. This result suggests that the (His)3 ligand environment is essential for acceleration of the Cu-NiR reaction. The highest Cu-NiR activity for the Et-TIC complex can be explained by the structural and spectroscopic characterizations and the molecular orbital calculations presented in this paper. Based on these results, the functional role of the (His)3 ligand environment in Cu-NiR is discussed.

Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite Reductase by Small Molecules

The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

The enzyme mechanism of nitrite reductase studied at single-molecule level

A generic method is described for the fluorescence "readout" of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.

Role ofBradyrhizobium japonicumcytochromec550in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.