Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase.

Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-Å resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction.

Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation.

Heterodimeric 2-oxoacid:ferredoxin oxidoreductase (OFOR) from Sulfolobus tokodaii (StOFOR) has only one [4Fe-4S]²âº cluster, ligated by 4 Cys residues, C12, C15, C46, and C197. The enzyme has no other Cys. To elucidate the role of these Cys residues in holding of the iron-sulfur cluster in the course of oxidative decarboxylation of a 2-oxoacid, one or two of these Cys residues was/were substituted with Ala to yield C12A, C15A, C46A, C197A and C12/15A mutants. All the mutants showed the loss of iron-sulfur cluster, except the C197A one which retained some unidentified type of iron-sulfur cluster. On addition of pyruvate to OFOR, the wild type enzyme exhibited a chromophore at 320nm and a stable large EPR signal corresponding to a hydroxyethyl-ThDP radical, while the mutant enzymes did not show formation of any radical intermediate or production of acetyl-CoA, suggesting that the intact [4Fe-4S] cluster is necessary for these processes. The stable radical intermediate in wild type OFOR was rapidly decomposed upon addition of CoA in the absence of an electron acceptor. Non-oxidative decarboxylation of pyruvate, yielding acetaldehyde, has been reported to require CoA for other OFORs, but StOFOR catalyzed acetaldehyde production from pyruvate independent of CoA, regardless of whether the iron-sulfur cluster is intact [4Fe-4S] type or not. A comprehensive reaction scheme for StOFOR with a single cluster was proposed.

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.

Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.

Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 µM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family.

Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum.

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-ß1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 ß-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-ß1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique ß-trefoil-like fold. Compared with other ß-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for ß-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near (4)E, whereas that of LNB-thiazoline is in a (4)C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Crystal structure of a feruloyl esterase belonging to the tannase family: a disulfide bond near a catalytic triad.

Feruloyl esterase (FAE) catalyzes the hydrolysis of the ferulic and diferulic acids present in plant cell wall polysaccharides, and tannase catalyzes the hydrolysis of tannins to release gallic acid. The fungal tannase family in the ESTHER database contains various enzymes, including FAEs and tannases. Despite the importance of FAEs and tannases in bioindustrial applications, three-dimensional structures of the fungal tannase family members have been unknown. Here, we determined the crystal structure of FAE B from Aspergillus oryzae (AoFaeB), which belongs to the fungal tannase family, at 1.5 Å resolution. AoFaeB consists of a catalytic α/ß-hydrolase fold domain and a large lid domain, and the latter has a novel fold. To estimate probable binding models of substrates in AoFaeB, an automated docking analysis was performed. In the active site pocket of AoFaeB, residues responsible for the substrate specificity of the FAE activity were identified. The catalytic triad of AoFaeB comprises Ser203, Asp417, and His457, and the serine and histidine residues are directly connected by a disulfide bond of the neighboring cysteine residues, Cys202 and Cys458. This structural feature, the "CS-D-HC motif," is unprecedented in serine hydrolases. A mutational analysis indicated that the novel structural motif plays essential roles in the function of the active site.

Crystal structure of glycoside hydrolase family 127 ß-l-arabinofuranosidase from Bifidobacterium longum.

Enzymes acting on ß-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of ß-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a ß-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked ß-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and ß-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional ß-sandwich domains, with one ß-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn(2+) coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the ß-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

Sulfolobus tokodaii ST2133 is characterized as a thioredoxin reductase-like ferredoxin:NADP+ oxidoreductase.

The putative gene (st2133) for ferredoxin:NADP(+) oxidoreductase (FNR) from Sulfolobus tokodaii, a thermoacidophilic crenarchaeon, was heterologously expressed. About 90% of the purified product was a homodimer containing 0.46 mol FAD/mol subunit, and showing NADPH:DCPIP oxidoreductase activity, V max being 1.38 and 21.8 U/mg (70 °C) in the absence and presence of 1 mM FMN. NADPH was a much better electron donor than NADH with various electron acceptors, such as oxygen, hydrogen peroxide, DCPIP, cytochrome c, and dithiobisnitrobenzoate. Most of the reactions were activated by 15- to 140-fold on addition of FMN, while FAD was 5-10 times less effective. Ferredoxin (Fd) from S. tokodaii served as an electron carrier in both Fd-dependent NADPH formation and NADPH-dependent Fd reduction. ST2133 belongs to the thioredoxin reductase-like protein family, which is slightly distantly related to FNR family proteins from bacteria, plants and man. This is the first report on FNR from a crenarchaeon, providing a clue to the recycling of Fd during archaeal metabolism.

Crystal structures of glycoside hydrolase family 3 ß-glucosidase 1 from Aspergillus aculeatus.

GH3 (glycoside hydrolase family 3) BGLs (ß-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

Archaeal aldehyde dehydrogenase ST0064 from Sulfolobus tokodaii, a paralog of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, is a succinate semialdehyde dehydrogenase.

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (Km of 0.0029 mM and kcat of 30.0 s(-1)) with no allosteric regulation. Activity toward GAP was lower (Km of 4.6 mM and kcat of 4.77 s(-1)), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.

Advenella faeciporci sp. nov., a nitrite-denitrifying bacterium isolated from nitrifying-denitrifying activated sludge collected from a laboratory-scale bioreactor treating piggery wastewater.

Strain M-07(T) was isolated from nitrifying-denitrifying activated sludge treating piggery wastewater. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain M-07(T) belonged to the genus Advenella. 16S rRNA gene sequence similarity between M-07(T) and Advenella incenata CCUG 45225(T), Advenella mimigardefordensis DPN7(T) and Advenella kashmirensis WT001(T) was 96.5, 97.3 and 96.9%, respectively. The DNA G+C content of strain M-07(T) was 49.5 mol%, which was approximately 5 mol% lower than the range for the genus Advenella (53.5-58.0 mol%). The predominant cellular fatty acids of strain M-07(T) were C(16:0), summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(17:0) cyclo and summed feature 2 (comprising one or more of C(14:0) 3-OH, iso-C(16:1) I, an unidentified fatty acid with an equivalent chain-length of 10.928 and C(12:0) alde). The isoprenoid quinone was Q-8. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness, strain M-07(T) should be classified as a novel species of the genus Advenella, for which the name Advenella faeciporci sp. nov. is proposed. The type strain is M-07(T) ( = JCM 17746(T)  = KCTC 23732(T)).

Crystal structures of glycoside hydrolase family 51 α-L-arabinofuranosidase from Thermotoga maritima.

α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-ß-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8-2.3 Å resolution to determine the architecture of the substrate binding pocket. Subsite -1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.

Regio- and stereospecificity of filipin hydroxylation sites revealed by crystal structures of cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis.

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the "filipin complex." CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1', respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B' helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.

Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,ß-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae.

Multiple flavohemoglobin (FHb) homolog genes are found in the genomes of eukaryotic microorganisms, but their functions remain unknown. In this study, two distinct types of FHbs (predictive cytosolic FHb1 and predictive mitochondrial FHb2) from the fungus Aspergillus oryzae were investigated to elucidate the physiological roles of these FHbs. The fhb1 gene responded to external nitric oxide (NO) stress at the transcriptional level, whereas the fhb2 gene did not. Disrupting fhb1 increased cell hypersensitivity to NO stress, whereas deficiency of the fhb2 gene had no effect on phenotype compared to the wild-type strain. By fusing GFP protein to FHbs, we determined that FHb1 and FHb2 are located in the cytosol and mitochondria, respectively. In the wild-type strain, the transcriptional level of the fhb2 gene was too low to be detected, but its expression was detectable in the NirK (mitochondrial copper-containing dissimilatory nitrite reductase) overexpression strain (AoHnirK), which showed a significantly higher denitrification capability than that shown by the wild-type strain. The induction of the fhb2 gene in the AoHnirK strain may be due to the abundance of NO produced by overexpressed NirK in the mitochondria. These results suggest that FHb1 and FHb2 may play a role in protecting cells from external and internal NO stress, respectively.

Conserved residues in membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon.

A membrane-intrinsic acid pyrophosphatase (ST2226) from Sulfolobus tokodaii, a thermoacidophilic archaeon, is possibly involved in glycoprotein biosynthesis and belongs to the phosphatidic acid phosphatase class 2 superfamily, including both membrane-intrinsic and soluble enzymes with divergent functions ranging from dephosphorylation of undecaprenylpyrophosphate and phospho-monoesters such as glucose-6-phosphate to vanadium-containing chloroperoxidation. ST2226 is an archaeal ortholog of these enzymes sharing a common phosphatase motif. Through site-directed mutagenesis as to each of the conserved residues, the catalytic roles of the latter were deduced, as well as the transmembrane topology with all the conserved residues in close proximity to the outside of the membrane.

Identification of the lysine residue responsible for coenzyme A binding in the heterodimeric 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii, a thermoacidophilic archaeon, using 4-fluoro-7-nitrobenzofurazan as an affinity label.

The heterodimeric 2-oxoacid:ferredoxin oxidoreductase (StOFOR) from Sulfolobus tokodaii, a thermoacidophilic archaeon, was inactivated by low concentrations of 4-fluoro-7-nitrobenzofurazan (NBD-F), with concomitant increase in fluorescence in subunit-b. The inactivation was prevented by CoA, suggesting that NBD-F covalently bound to the Lys which is responsible for CoA binding. The NBD-labeled subunit-b was isolated and digested with endoproteinase Lys-C. The resulting polypeptide mixture was separated by reverse phase HPLC and the fluorescent fraction was isolated. Amino acid sequencing of the fraction revealed that it comprised a mixture of two polypeptides containing Lys125 and Lys173, respectively. Two StOFOR mutants, K125A and K173A, were constructed, expressed and purified. K125A showed a large increase in the K(m) value for CoA and showed poor inactivation by NBD-F, compared with K173A and wild type StOFOR, indicating Lys125 in subunit-b is the critical residue that interacts with CoA.

Aspergillus oryzae flavohemoglobins promote oxidative damage by hydrogen peroxide.

Apart from their well-established role in nitric oxide detoxification, flavohemoglobins (FHbs) are also believed to be involved in protection against oxidative stress in some yeast and bacteria. However, different studies have reported contradictory results in this regard. Here, we investigate the relationship between two FHbs in Aspergillus oryzae (cytosolic FHb1 and mitochondrial FHb2) and oxidative stress. The strains deficient in the two FHbs exhibited higher resistance to hydrogen peroxide than the wild-type. In addition, the FHb2 overexpression strain showed hypersensitivity to hydrogen peroxide. Flavin reductase accompanied by the ferric reductase activities of the two FHbs were observed in correspondence with this expression. The reductase activities of the FHbs were attributed to their C-terminal flavin reductase domains. The reduced intracellular free iron can subsequently promote oxidative damage by accelerating the Fenton reaction in the cytosol and mitochondria (corresponding to the subcellular localizations of the two FHbs). This study is the first to show that fungal FHbs have a deleterious effect on oxidative protection, and suggests that the accelerated Fenton reaction induced by FHbs might be responsible for this effect.

Potential of aerobic denitrification by Pseudomonas stutzeri TR2 to reduce nitrous oxide emissions from wastewater treatment plants.

In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N(2)O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N(2)O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N(2)O were present, strain TR2 reduced N(2)O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd(1) nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N(2)O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N(2)O emissions when applied to sewage disposal fields.