Effects of gaseous NO2 on cells of Nitrosomonas eutropha previously incapable of using ammonia as an energy source

Cells of Nitrosomonas eutropha grown under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were initially unable to oxidize ammonia (ammonium) and hydroxylamine when transferred to oxic conditions. Recovery of ammonia and hydroxylamine oxidation activity was dependent on the presence of NO₂. Under oxic conditions, without addition of NO₂, ammonia consumption started after 8 – 9 days, and small amounts of NO and NO₂ were detectable in the gas atmosphere. Removing these nitrogen oxides by intensive aeration, ammonia oxidation activity decreased and broke off after 15 days. Addition of gaseous NO₂ (25 ppm) led to a fast recovery of ammonia oxidation (3 days). Simultaneously, the arrangement of intracytoplasmic membranes (ICM) changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentration of a 27 and a 30 kDa polypeptide, and the cytochrome c content increased significantly.     

14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase.

Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27-kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase.     

Tri- and tetramethylhydroquinone as electron donors for ammonia monooxygenase in whole cells of Nitrosomonas europaea

Abstract Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole cells of Nitrosomonas europaea . Positive results were obtained with tri- and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethylphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with tri-and tetramethylhydroquinone were 1.8 and 4.6 μmol · h⁻¹· mg protein⁻¹, respectively.     

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.     

Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.     

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.

The effect of copper on the in vivo and in vitro activity of ammonia monooxygenase (AMO) from the nitrifying bacterium Nitrosomonas europaea was investigated. The addition of CuCl2 to cell extracts resulted in 5- to 15-fold stimulation of ammonia-dependent O2 consumption, ammonia-dependent nitrite production, and hydrazine-dependent ethane oxidation. AMO activity was further stimulated in vitro by the presence of stabilizing agents, including serum albumins, spermine, or MgCl2. In contrast, the addition of CuCl2 and stabilizing agents to whole-cell suspensions did not result in any stimulation of AMO activity. The use of the AMO-specific suicide substrate acetylene revealed two populations of AMO in cell extracts. The low, copper-independent (residual) AMO activity was completely inactivated by acetylene in the absence of exogenously added copper. In contrast, the copper-dependent (activable) AMO activity was protected against acetylene inactivation in the absence of copper. However, in the presence of copper both populations of AMO were inactivated by acetylene. [14C]acetylene labelling of the 27-kDa polypeptide of AMO revealed the same extent of label incorporation in both whole cells and optimally copper-stimulated cell extracts. In the absence of copper, the label incorporation in cell extracts was proportional to the level of residual AMO activity. Other metal ions tested, including Zn2+, Co2+, Ni2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, and Ag+, were ineffective at stimulating AMO activity or facilitating the incorporation of 14C label from [14C]acetylene into the 27-kDa polypeptide. On the basis of these results, we propose that loss of AMO activity upon lysis of N. europaea results from the loss of copper from AMO, generating a catalytically inactive, yet stable and activable, form of the enzyme.     

Oxidation of methyl fluoride and dimethyl ether by ammonia monooxygenase in Nitrosomonas europaea.

Methyl fluoride and dimethyl ether were previously identified as inhibitors of ammonia oxidation and N2O production in autotrophic nitrifying bacteria. We demonstrate that methyl fluoride and dimethyl ether are substrates for ammonia monooxygenase and are converted to formaldehyde and a mixture of methanol and formaldehyde, respectively.     

Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea

The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level.     

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.