Copper-containing Nitrite Reductase Research Articles

Genome of a Novel Bacterium "Candidatus Jettenia ecosi" Reconstructed From the Metagenome of an Anammox Bioreactor.

The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria “Candidatus Jettenia ecosi” developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, “Candidatus Jettenia ecosi” to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of “Candidatus Jettenia ecosi” revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.

Proton-Coupled-Electron Transfer Enhances the Electrocatalytic Reduction of Nitrite to NO in a Bioinspired Copper Complex

The selective and efficient electrocatalytic reduction of nitrite to nitric oxide (NO) is of tremendous importance, both for the development of NO-release systems for biomedical applications and for the removal of nitrogen oxide pollutants from the environment. In nature, this transformation is mediated by (amongst others) enzymes known as the copper-containing nitrite reductases. The development of synthetic copper complexes that can reduce nitrite to NO has therefore attracted considerable interest. However, there are no studies describing the crucial role of proton-coupled-electron transfer during nitrite reduction when using such synthetic complexes. In this talk, I will describe a system for the electrocatalytic reduction of nitrite to NO in which the role of proton-relaying units in the secondary coordination sphere of the metal can be probed for the first time (ACS Catal. 2018, 8, 5070–5084). Our results suggest that positioning a pendant carboxylate group in close proximity to the copper centre gives an electrocatalyst that is twice as effective for nitrite reduction than the analogous compound that lacks such a proton-relaying moiety. These results highlight the critical role of proton-coupled-electron transfer in the reduction of nitrite to NO and have important implications for the design of biomimetic catalysts for the selective interconversions of the nitrogen oxides. Figure 1

Depth distributions of nitrite reductase (nirK) gene variants reveal spatial dynamics of thaumarchaeal ecotype populations in coastal Monterey Bay.

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are key players in nutrient cycling, yet large gaps remain in our understanding of their ecology and metabolism. Despite multiple lines of evidence pointing to a central role for copper-containing nitrite reductase (NirK) in AOA metabolism, the thaumarchaeal nirK gene is rarely studied in the environment. In this study, we examine the diversity of nirK in the marine pelagic environment, in light of previously described ecological patterns of pelagic thaumarchaeal populations. Phylogenetic analyses show that nirK better resolves diversification patterns of marine Thaumarchaeota, compared to the conventionally used marker gene amoA. Specifically, we demonstrate that the three major phylogenetic clusters of marine nirK correspond to the three 'ecotype' populations of pelagic Thaumarchaeota. In this context, we further examine the relative distributions of the three variant groups in metagenomes and metatranscriptomes representing two depth profiles in coastal Monterey Bay. Our results reveal that nirK effectively tracks the dynamics of thaumarchaeal ecotype populations, particularly finer-scale diversification patterns within major lineages. We also find evidence for multiple copies of nirK per genome in a fraction of thaumarchaeal cells in the water column, which must be taken into account when using it as a molecular marker.

Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography.

Copper-containing nitrite reductases (CuNiRs) that convert NO2 - to NO via a CuCAT-His-Cys-CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD-, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2 - a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis.

It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.

A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase.

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2−) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2− to the T2Cu centre, but very little is known about how NO2− binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2− may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2− with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

Crystallographic study of dioxygen chemistry in a copper-containing nitrite reductase from Geobacillus thermodenitrificans.

Copper-containing nitrite reductases (CuNIRs) are multifunctional enzymes that catalyse the one-electron reduction of nitrite (NO2-) to nitric oxide (NO) and the two-electron reduction of dioxygen (O2) to hydrogen peroxide (H2O2). In contrast to the mechanism of nitrite reduction, that of dioxygen reduction is poorly understood. Here, results from anaerobic synchrotron-radiation crystallography (SRX) and aerobic in-house radiation crystallography (iHRX) with a CuNIR from the thermophile Geobacillus thermodenitrificans (GtNIR) support the hypothesis that the dioxygen present in an aerobically manipulated crystal can bind to the catalytic type 2 copper (T2Cu) site of GtNIR during SRX experiments. The anaerobic SRX structure showed a dual conformation of one water molecule as an axial ligand in the T2Cu site, while previous aerobic SRX GtNIR structures were refined as diatomic molecule-bound states. Moreover, an SRX structure of the C135A mutant of GtNIR with peroxide bound to the T2Cu atom was determined. The peroxide molecule was mainly observed in a side-on binding manner, with a possible minor end-on conformation. The structures provide insights into dioxygen chemistry in CuNIRs and hence help to unmask the other face of CuNIRs.

Solution Structure Determination of a Copper-containing Nitrite Reductase of Achromobacter cycloclastes by Small Angle Neutron Scattering

A copper-containing nitrite reductase (NiR) catalyzes NO2- reduction in denitrifying bacteria. The reaction mechanism was proposed based on the trimeric crystal structure. In this study, a small angle neutron scattering (SANS) experiment of NiR from Achromobacter cycloclastes (AcNiR) was performed, and the first solution structure was obtained. The obtained radius of gyration, the calculation of the scattering curve from the available pdb structure and Ab Initio modelling all indicate a trimeric structure of AcNiR in solution.

Proton-Coupled Electron Transfer Enhances the Electrocatalytic Reduction of Nitrite to NO in a Bioinspired Copper Complex

The selective and efficient electrocatalytic reduction of nitrite to nitric oxide (NO) is of tremendous importance, both for the development of NO-release systems for biomedical applications and for the removal of nitrogen oxide pollutants from the environment. In nature, this transformation is mediated by (among others) enzymes known as the copper-containing nitrite reductases. The development of synthetic copper complexes that can reduce nitrite to NO has therefore attracted considerable interest. However, there are no studies describing the crucial role of proton-coupled electron transfer during nitrite reduction when such synthetic complexes are used. Herein, we describe the synthesis and characterization of two previously unreported Cu complexes (3 and 4) for the electrocatalytic reduction of nitrite to NO, in which the role of proton-relaying units in the secondary coordination sphere of the metal can be probed. Complex 4 bears a pendant carboxylate group in close proximity to the copper center, whi...

Redundant roles of Bradyrhizobium oligotrophicum Cu-type (NirK) and cd1-type (NirS) nitrite reductase genes under denitrifying conditions.

Reduction of nitrite to nitric oxide gas by respiratory nitrite reductases (NiRs) is the key step of denitrification. Denitrifiers are strictly divided into two functional groups based on whether they possess the copper-containing nitrite reductase (CuNiR) encoded by nirK or the cytochrome cd1 nitrite reductase (cdNiR) encoded by nirS. Recently, some organisms carrying both nirK and nirS genes have been found. Bradyrhizobium oligotrophicum S58 is a nitrogen-fixing oligotrophic bacterium that carries a set of genes for complete denitrification of nitrate to dinitrogen, including nirK and nirS genes. We show that denitrification in S58 is functional under low-oxygen conditions (anaerobiosis and microaerobiosis), but not under aerobiosis. Under denitrifying conditions, the ΔnirK and ΔnirS single S58 mutants grew normally and their NiR activity was not affected. However, the ΔnirKS double mutant grew more slowly, presumably because the impaired NiR activity resulted in nitrite accumulation in the medium. These results suggest a redundant role for nirK and nirS genes in B. oligotrophicum S58 denitrification. In addition, we found that the nirS gene product, but not that of nirK, maintains swimming motility of S58 under aerobic and low-oxygen conditions in the presence of nitrate.

Effect of Nitrate Amendment on Soil Denitrification Activity and Anthracene Anaerobic Degradation

The degradation of soil polycyclic aromatic hydrocarbons (PAHs) under denitrification is one of the most important pathways for anaerobic PAH elimination, but little is known about the effect of nitrate (the terminal electron acceptor for denitrification) on soil denitrification activity and PAH degradation under anaerobic conditions. In this study, the effect of nitrate on soil anthracene anaerobic degradation and denitrification activity was investigated through an anaerobic microcosm experiment. Two groups of treatments without (N0) and with (N30) nitrate (30 mg·kg-1) amendment were conducted. Each group contained three treatments with different anthracene concentrations (0, 15, and 30 mg·kg-1, denoted as A0, A15, and A30, respectively). Therefore, a total of six treatments (N0A0, N0A15, N0A30, N30A0, N30A15, and N30A30) were incubated in darkness at 25℃ for 45 days, and the production rates of N2O and CO2, abundances of denitrification related genes (narG:periplasmic nitrate reductase gene; nirK:copper-containing nitrite reductase gene; and nirS:cd1-nitrite reductase gene), and soil anthracene content were measured at 3, 7, 14, and 45 days. The results indicated that the intensive denitrification enzyme activity in each treatment was only detected at day 3, which could be significantly enhanced by both nitrate and anthracene amendments. Subsequently, a sharp decline of denitrification enzyme activity was observed in each treatment, while anthracene showed an obvious inhibition of soil denitrification enzyme activity. The result of a two-way ANOVA also indicated that nitrate, anthracene, and their interactions had significant effects on soil denitrification enzyme activity. The result of a quantitative-PCR indicated that, during the incubation, the abundances of narG and nirS exhibited an increasing tendency, but the abundance of nirK was relatively constant compared with its former counterparts. The final removal rate of anthracene under anaerobic soil environment was in the range of 33.83%-55.01%, and neither the final removal rate nor the degradation rate of anthracene could be significantly affected by nitrate amendment during incubation. The anthracene degradation rates in the higher anthracene containing treatments (N0A30 and N30A30) were significantly higher than those in the lower anthracene containing treatments (N0A15 and N30A15). In summary, nitrate amendments had no effect on soil anthracene anaerobic degradation but could significantly affect soil denitrification enzyme activity and the abundance of denitrification related narG and nirS genes.

Intra-electron transfer induced by protonation in copper-containing nitrite reductase.

The inter- and intra-electron and proton transfers in the nitrite reduction of copper-containing nitrite reductase (CuNiR) were investigated by using the QM/MM method with the calculational models containing type 1 (T1) and type 2 (T2) Cu sites. The electron transfer from the outer electron donor protein to the T1 Cu site occurred both before and after nitrite binding, and nitrite binding lowered the reduction potential of the Cu T1 site. The protonation of catalytic His244 subsequent to nitrite binding and T1 Cu reduction induced partial intra-electron transfer from T1 to T2 Cu sites. The proton transfer from His244 to nitrite bound on the T2 Cu site via the hydrogen bond network induced intra-electron transfer from the T1 to T2 Cu site. The interaction of the T1 Cu ligand with the second sphere amino acid residues and water molecules affected the reduction potential of the T1 Cu site. The water molecules in the so-called proton pool have an important role in the regulation of the basicity of His244. The conformation of the sensor loop did not change along the reaction, but the water molecule network extending along the sensor loop was changed by nitrite binding.

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s−1) lower than that of wt SmNir (kcat=240(50) s−1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the Nδ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway.

Nitrogen removal during the cold season by constructed floating wetlands planted with Oenanthe javanica

Constructed floating wetlands (CFWs) are used to treat waste waters of various origins either alone or as part of waste water treatment trains. The aim of the present study was to determine the extent of nitrogen removal by CFWs planted with Oenanthe javanica (Blume) DC. at low temperatures (<10°C) and whether CFWs with vesuvianite as a substrate perform better than those without substrate. A batch model was used, with CFWs planted with O. javanica (Tc), CFWs without O. javanica (Ts), CFWs without substrate (Tp) and floating mats only (To) as a control. The average removal rates of NH4+-N, NO3–-N and total nitrogen were 78.3, 44.4 and 49.7% respectively in Tc; 72.0, 40.0 and 39.5% respectively in Ts; and 73.1, 33.7 and 44.0% respectively in Tp. In addition to a gradual increase in chemical oxygen demand during the experimental period, Tc had higher microbial richness and diversity, as well as a higher abundance of bacteria, archaea, anaerobic ammonium oxidation (Anammox) bacteria and key genes (ammonia mono-oxygenase, amoA, nitrous oxide reductase, nosZ, dissimilatory cd1-containing nitrite reductase, nirS, and dissimilatory copper-containing nitrite reductase, nirK) involved in nitrogen metabolism in the substrate than Ts. Further analysis of microbial community composition revealed a difference at multiple taxonomic levels among different systems. These results demonstrate the positive roles of O. javanica and vesuvianite in CFWs in nitrogen removal from waste water during the cold season (mean water temperature <10°C).

Copper-Containing Nitrite Reductase Employing Proton-Coupled Spin-Exchanged Electron-Transfer and Multiproton Synchronized Transfer to Reduce Nitrite.

The possible catalytic mechanism of the reduction of nitrite by copper-containing nitrite reductases (CuNiRs) is examined by using the M06 function according to two copper models, which include type-one copper (T1Cu) and type-two copper (T2Cu) sites. Examinations confirm that the protonation of two residues, His255 and Asp98, near the T2Cu site, can modulate the redox states of T1Cu and T2Cu, but cannot directly cause electron transfer from T1Cu to T2Cu. The electron hole remains at the T2Cu site when only one residue, His255 or Asp98, is protonated. However, the hole resides at the T1Cu site when both His255 and Asp98 are protonated. Then, the first protonation of nitrite takes place through indirect proton transfer from protonated His255 through the bridging H2 O and Asp98 with three protons moving together, which cannot cause the cleavage of the HO-NO bond. Subsequently, the substrate is required to obtain another proton from reprotonated His255 through the bridging H2 O. The reprotonation of nitrite induces the generation of nitric oxide (NO) and H2 O at the T2Cu site through a special double-proton-coupled spin-exchanged electron-transfer mechanism with indirect proton transfer from His255 to the substrate, a beta-electron of T2CuI shift to the NO cation, and the remaining alpha-electron changing spin direction at the same time. These results may provide useful information to better understand detailed proton-/electron-transfer reactions for the catalytic processes of CuNiR.

Potential for aerobic NO2- reduction and corresponding key enzyme genes involved in Alcaligenes faecalis strain NR.

The potential for aerobic NO2- removal by Alcaligenes faecalis strain NR was investigated. 35mg/L of NO2--N was removed by strain NR under aerobic conditions in the presence of NH4+. 15N-labeling experiment demonstrated that N2O and N2 were possible products during the aerobic nitrite removal process by strain NR. The key enzyme genes of nirK, norB and nosZ, which regulate the aerobic nitrite denitrification process, were successfully amplified from strain NR. The gene sequence analysis indicates that copper-containing nitrite reductase (NIRK) and periplasmic nitrous oxide reductase (NOSZ) were both hydrophilic protein and the transmembrane structures were absent, while nitric oxide reductase large subunit (NORB) was a hydrophobic and transmembrane protein. According to the three-dimensional structure and binding site analysis, the bulky and hydrophobic methionine residue proximity to the nitrite binding sites of NIRK was speculated to be related to the oxygen tolerance of NIRK from strain NR.

An investigation into the unusual linkage isomerization and nitrite reduction activity of a novel tris(2-pyridyl) copper complex.

The copper-containing nitrite reductases (CuNIRs) are a class of enzymes that mediate the reduction of nitrite to nitric oxide in biological systems. Metal–ligand complexes that reproduce the salient features of the active site of CuNIRs are therefore of fundamental interest, both for elucidating the possible mode of action of the enzymes and for developing biomimetic catalysts for nitrite reduction. Herein, we describe the synthesis and characterization of a new tris(2-pyridyl) copper complex ([Cu1(NO2)2]) that binds two molecules of nitrite, and displays all three of the common binding modes for , with one nitrite bound in an asymmetric quasi-bidentate κ2-ONO manner and the other bound in a monodentate fashion with a linkage isomerism between the κ1-ONO and κ1-NO2 binding modes. We use density functional theory to help rationalize the presence of all three of these linkage isomers in one compound, before assessing the redox activity of [Cu1(NO2)2]. These latter studies show that the complex is not a competent nitrite reduction electrocatalyst in non-aqueous solvent, even in the presence of additional proton donors, a finding which may have implications for the design of biomimetic catalysts for nitrite reduction.

Co-effects of pyrene and nitrate on the activity and abundance of soil denitrifiers under anaerobic condition.

It has previously been confirmed that polycyclic aromatic hydrocarbons (PAHs) could be degraded by soil microbes coupling with denitrification, but the relationships among soil denitrifiers, PAHs, and nitrate under obligate anaerobic condition are still unclear. Here, co-effects of pyrene and nitrate on the activity and abundance of soil denitrifiers were investigated through a 45-day incubation experiment. Two groups of soil treatments with (N30) and without (N0) nitrate (30mgkg-1 dry soil) amendment were conducted, and each group contained three treatments with different pyrene concentrations (0, 30, and 60mgkg-1 dry soil denoted as P0, P30, and P60, respectively). The pyrene content, abundances of denitrification concerning genes (narG, periplasmic nitrate reductase gene; nirS, cd 1-nitrite reductase gene; nirK, copper-containing nitrite reductase gene), and productions of N2O and CO2 were measured at day 3, 14, 28, and 45, and the bacterial community structures in four represented treatments (N0P0, N0P60, N30P0, and N30P60) were analyzed at day 45. The results indicated that the treatments with higher pyrene concentration had higher final pyrene removal rates than the treatments with lower pyrene concentration. Additionally, intensive emission of N2O was detected in all treatments only at day 3, but a continuous production of CO2 was measured in each treatment during the incubation. Nitrate amendment could enhance the activity of soil denitrifiers, and be helpful for soil microbes to sustain their activity. While pyrene seemed had no influence on the productions of N2O and CO2, and amendment with pyrene or nitrate both had no obvious effect on abundances of denitrification concerning genes. Furthermore, it was nitrate but not pyrene had an obvious influence on the community structure of soil bacteria. These results revealed that, under anaerobic condition, the activity and abundance of soil denitrifiers both were insensitive to pyrene, but nitrate could improve the activity of soil denitrfiers and induce the shifts in soil bacterial community structure.

New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper.

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis (AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz.

Variations in abundance and community composition of denitrifying bacteria during a cyanobacterial bloom in a eutrophic shallow lake in China

ABSTRACTAlthough cyanobacterial blooms can change microbial communities, it is still unclear what impact such harmful blooms will have on denitrifying bacteria, the drivers of the removal of excessive nitrogen from water. In order to clarify the impact, populations of denitrifying bacteria, with periodic proliferation and dominance of cyanobacteria in a eutrophic shallow lake located in southeast China, were investigated using quantitative real-time polymerase chain reaction (qPCR) and 454-pyrosequencing based on the copper-containing nitrite reductase (nirK) gene, cytochrome cd1-containing nitrite reductase (nirS) gene and nitrous oxide reductase (nosZ) gene. Samples were collected periodically during a three-month period when the cyanobacterial density gradually increased. In the qPCR analyses, abundances of nirK, nirS and nosZ were intensely positively correlated with the biomass of cyanobacteria. Moreover, 454-pyrosequencing revealed that the community composition of denitrifying bacteria shifted with the increase in cyanobacterial density. These results indicated that the shifts of the community composition of denitrifying bacteria might be related to cyanobacterial blooms, which could potentially lead to alterations of denitrification in eutrophic water.