Novel nirK cluster genes in Nitrosomonas europaea are required for NirK-dependent tolerance to nitrite

Nitrite reductase (NirK) of Nitrosomonas europaea confers tolerance to nitrite (NO2-). The nirK gene is clustered with three genes of unknown physiological function: ncgABC. At present, this organization is unique to nitrifying bacteria. Here we report that the ncgABC gene products facilitate NirK-dependent NO2- tolerance by reversing the negative physiological effect that is associated with the activity of NirK in their absence. We hypothesize that the ncg gene products are involved in the detoxification of nitric oxide that is produced by NirK.     

The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing ;denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO(2) (-) or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described.     

Transcriptome of a Nitrosomonas europaea mutant with a disrupted nitrite reductase gene (nirK)

Global gene expression was compared between the Nitrosomonas europaea wild type and a nitrite reductase-deficient mutant using a genomic microarray. Forty-one genes were differentially regulated between the wild type and the nirK mutant, including the nirK operon, genes for cytochrome c oxidase, and seven iron uptake genes. Relationships of differentially regulated genes to the nirK mutant phenotype are discussed.     

Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism. The loss of activity was specific to the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH₄⁺-dependent O₂ consumption, some loss of active AMO molecules observed by polypeptide labeling with ¹⁴C₂H₂, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.     

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2 weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24 h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.     

Distribution of Nitrosomonas europaea and Paracoccus denitrificans immobilized in tubular polymeric gel for nitrogen removal

To improve the cooperative removal of nitrogen by Nitrosomonas europaea and Paracoccus denitrificans, we controlled their distribution in a tubular gel. When ethanol was supplied inside the tubular gel as an electron donor, their distributions overlapped in the external region of the gel. By changing the electron donor from ethanol to gaseous hydrogen, the distribution of P. denitrificans shifted to the inside of the tube and was separated from that of N. europaea. The separation resulted in an increase of the oxidation rate of ammonia by 25%.     

Nitrite metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrite uptake and nitrite reductase by ammonia

Abstract Nitrogen regulation of nitrite uptake and nitrite reductase was studied in the cyanobacterium Anabaena cycadeae and its glutamine-auxotrophic mutant. The development of the nitrite-uptake system preceded, and was independent of, the development of nitrate reductase. The levels of both of the systems were higher in the glutamine auxotroph lacking glutamine synthetase (GS) than in the wild-type strain having normal GS activity. The nitrite-uptake system was found to be constitutive and ammonia-repressible whereas the nitrite-reductase system was ammonia-repressible and nitrite-inducible. Ammonia did not inhibit the nitrite-uptake and nitrite reductase activities in the glutamine auxotroph whereas glutamine did so, suggesting that repression of nitrite-uptake and nitrite reductase systems by ammonia requires the operation of GS and probably involves the participation of some organic nitrogen metabolites like glutamine.     

Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.     

Poplar metal tolerance protein 1 confers zinc tolerance and is an oligomeric vacuolar zinc transporter with an essential leucine zipper motif

Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. Here, we describe the identification, characterization, and localization of PtdMTP1, a member of the CDF family from the hybrid poplar Populus trichocarpa x Populus deltoides. PtdMTP1 is expressed constitutively and ubiquitously, although at low levels. Heterologous expression in yeast showed that PtdMTP1 was able to complement the hypersensitivity of mutant strains to Zn but not to other metals, including Cd, Co, Mn, and Ni. PtdMTP1 fused to green fluorescent protein localized to the vacuolar membrane both in yeast and in plant cells, consistent with a function of PtdMTP1 in zinc sequestration. Overexpression of PtdMTP1 in Arabidopsis confers Zn tolerance. We show that PtdMTP1, when expressed in yeast and Arabidopsis, forms homooligomers, a novel feature of CDF members. Oligomer formation is disrupted by reducing agents, indicating possible disulfide bridge formation. PtdMTP1 also contains a conserved Leu zipper motif. Although not necessary for oligomer formation, Leu residues within this motif are required for PtdMTP1 functional activity.     

A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.     

Ammonia and O2 uptake in relation to proton translocation in cells of Nitrosomonas europaea

The uptake of ammonia and O₂ by washed cells of Nitrosomonas has been followed simultaneously and continuously using electrode techniques. The stoichiometry of NH ₄ ⁺ oxidation, O₂ uptake and NO ₂ ^- production was 1 : 1.5 : 1.0 and for NH₂OH oxidation a ratio of 1 for O₂ : NO ₂ ^- . A variety of inhibitors of electron transport and metals as well as uncouplers restricted ammonia uptake more markedly than O₂ utilization. There is good evidence for the involvement of copper in the NH ₄ ⁺ uptake process.A quinacrine fluorescence technique has been used to study the proton extrusion by washed cells on adding NH₄Cl and NH₂OH respectively as substrates. The uptake of NH ₄ ⁺ was followed by the extrusion of H⁺ and this process was depressed by those inhibitors which were also effective in the electrode experiments. A requirement for copper is also established for the translocation of protons into the medium, resulting from the uptake of NH ₄ ⁺ by cells.Abbreviations mCCCP carbonyl cyanide m-chlorophenylhydrazone - DBP 2,4 dibromophenol - DCCD N-N-dicyclohexylcarbodimide - DIECA Sodium diethyldithiocarbamate - DNP 2,4 dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - NBD chloride 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - N-serve 2-chloro-6-trichloromethyl-pyridine - PCP pentachlorophenol - 2-TMP 2-trichloromethyl-pyridine - TPB tetraphenylboron - TTFA 1-[thenoyl-(2)]-3,3,3-trifluoracetone - KSCN Potassium thiocyanate     

Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces

Summary The membrane lipid composition of Saccharomyces was manipulated by growing cells anaerobically with or without ergosterol and unsaturated fatty acid. Cells low in ergosterol but enriched in unsaturated fatty acid residues on membrane phospholipids produced high concentrations, 13–15.5% w/v, of ethanol at substrate conversion efficiencies of around 90%.