scholarly journals Ammonium Limitation Results in the Loss of Ammonia-Oxidizing Activity in Nitrosomonas europaea

1998 ◽  
Vol 64 (4) ◽  
pp. 1514-1521 ◽  
Author(s):  
Lisa Y. Stein ◽  
Daniel J. Arp
Keyword(s):  
Active Site ◽  
Ammonia Oxidation ◽  
Nitrosomonas Europaea ◽  
Ammonium Concentration ◽  
Ammonia Monooxygenase ◽  
Oxidation Activity ◽  
Ph Values ◽  
Nitrite Production ◽  
Steady State Levels ◽  
Logarithmic Growth

ABSTRACT The effects of limiting concentrations of ammonium on the metabolic activity of Nitrosomonas europaea, an obligate ammonia-oxidizing soil bacterium, were investigated. Cells were harvested during late logarithmic growth and were incubated for 24 h in growth medium containing 0, 15, or 50 mM ammonium. The changes in nitrite production and the rates of ammonia- and hydroxylamine-dependent oxygen consumption were monitored. In incubations without ammonium, there was little change in the ammonia oxidation activity after 24 h. With 15 mM ammonium, an amount that was completely consumed, there was an 85% loss of the ammonia oxidation activity after 24 h. In contrast, there was only a 35% loss of the ammonia oxidation activity after 24 h in the presence of 50 mM ammonium, an amount that was not consumed to completion. There was little effect on the hydroxylamine oxidation activity in any of the incubations. The loss of ammonia oxidation activity was not due to differences in steady-state levels of ammonia monooxygenase (AMO) mRNA (amoA) or to degradation of the active site-containing subunit of AMO protein. The incubations were also conducted at a range of pH values to determine whether the loss of ammonia oxidation activity was correlated to the residual ammonium concentration. The loss of ammonia oxidation activity after 24 h was less at lower pH values (where the unoxidized ammonium concentration was higher). When added in conjunction with limiting ammonium, short-chain alkanes, which are alternative substrates for AMO, prevented the loss of ammonia oxidation activity at levels corresponding to their binding affinity for AMO. These results suggest that substrates of AMO can preserve the ammonia-oxidizing activity of N. europaea in batch incubations by protecting either AMO itself or other molecules associated with ammonia oxidation.

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

1998 ◽  
Vol 64 (10) ◽  
pp. 4098-4102 ◽  
Author(s):  
Lisa Y. Stein ◽  
Daniel J. Arp
Keyword(s):  
Ammonia Oxidation ◽  
Nitrosomonas Europaea ◽  
Ammonia Monooxygenase ◽  
Oxidation Activity ◽  
Monooxygenase Activity ◽  
Nitrite Toxicity ◽  
Acidic Conditions ◽  
Ammonia Oxidizing Bacterium ◽  
Aerobic And Anaerobic ◽  
Unique Mechanism

ABSTRACT 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 O2consumption, 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.

Chlorinated solvents cometabolism by an enriched nitrifying bacterial consortium

2001 ◽  
Vol 1 (4) ◽  
pp. 95-102
Author(s):  
P. Ginstet ◽  
J.M. Audic ◽  
J.C. Block
Keyword(s):  
Drinking Water ◽  
Active Site ◽  
Ammonia Oxidation ◽  
Competitive Inhibition ◽  
Chlorinated Solvents ◽  
Bacterial Consortium ◽  
Ammonia Monooxygenase ◽  
Batch Tests ◽  
Mixed Biomass

The biodegradability of three of the most frequently halogenated aliphatics (trichloroethene, chloroform and 1.1.1.-trichloroethane) found in drinking water aquifers by a nitrifying enriched mixed biomass was investigated during batch tests. Within this mixed biomass, ammonia oxidisers were the effective degraders. The presence of ammonia stimulated chlorocarbon biodegradation, and the presence of chlorocarbon inhibited ammonia oxidation. This contrasted phenomenon was explained by a balance between electron supply from ammonia necessary to sustain the chlorocarbon oxidation and competitive inhibition for the ammonia monooxygenase active site between both substrates. About 0.03 to 0.2% of the electrons generated by ammonia oxidation were used for chlorocarbon degradation. Trichloroethene and chloroform oxidation induced a biomass inactivation (around 30 to 40 mg of proteins inactivated per μmol of chlorocarbn oxidised). Biomass re-activation due to exergonic ammonia catabolism was estimated to 24±6 mg of proteins reactivated per mmol of ammonia oxidised in both cases. No inactivation of re-activation was observed in the case of 1.1.1-trichloroethane.

scholarly journals Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by Nitrosomonas europaea

1998 ◽  
Vol 64 (4) ◽  
pp. 1372-1378 ◽  
Author(s):  
Norman G. Hommes ◽  
Sterling A. Russell ◽  
Peter J. Bottomley ◽  
Daniel J. Arp
Keyword(s):  
Nitrosomonas Europaea ◽  
Exchange Capacity ◽  
Ammonium Concentration ◽  
Organic Carbon Content ◽  
Soil Slurry ◽  
Natural Systems ◽  
Exchangeable Acidity ◽  
Nitrite Production ◽  
Initial Ph ◽  
Substantial Drop

ABSTRACT Ammonia monooxygenase (AMO) from Nitrosomonas europaeacatalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of N. europaea. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with N. europaea cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by N. europaea. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH4 + in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH3 to concentrations (8 to 80 μM) similar to the Ks value of AMO for NH3 (∼29 μM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.

scholarly journals Inhibitory Effects of C2to C101-Alkynes on Ammonia Oxidation in Two Nitrososphaera Species

2015 ◽  
Vol 81 (6) ◽  
pp. 1942-1948 ◽  
Author(s):  
A. E. Taylor ◽  
K. Taylor ◽  
B. Tennigkeit ◽  
M. Palatinszky ◽  
M. Stieglmeier ◽  
...  
Keyword(s):  
Pure Culture ◽  
Ammonia Oxidation ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonia Monooxygenase ◽  
Inhibitory Effects ◽  
Content Type ◽  
Nitrite Production ◽  
Highly Sensitive ◽  
Production Activity ◽  
Ammonia Oxidizing Bacterium

ABSTRACTA previous study showed that ammonia oxidation by theThaumarchaeotaNitrosopumilus maritimus(group 1.1a) was resistant to concentrations of the C81-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C2to C101-alkynes were evaluated on the nitrite production activity of two pure culture isolates fromThaumarchaeotagroup 1.1b,Nitrososphaera viennensisstrain EN76 andNitrososphaera gargensis. BothN. viennensisandN. gargensiswere insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 μM) that did not inhibitN. maritimuspartially inhibited nitrite production inN. viennensisandN. gargensisin a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium,Nitrosomonas europaea, octyne inhibition ofN. viennensiswas: (i) fully and immediately reversible, (ii) not competitive with NH4+, and (iii) without effect on the competitive interaction between NH4+and acetylene. BothN. viennensisandN. gargensisdemonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed forN. maritimus, being highly sensitive to ≤C5alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed amongN. maritimus,N. viennensis, andN. gargensisin regard to the extent of their resistance/sensitivity to C6and C71-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among theThaumarchaeota.

scholarly journals Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis

2001 ◽  
Vol 67 (7) ◽  
pp. 2952-2957 ◽  
Author(s):  
Simon A. Q. Burton ◽  
Jim I. Prosser
Keyword(s):  
Ammonia Oxidation ◽  
Acid Soils ◽  
Urea Hydrolysis ◽  
Extracellular Ph ◽  
Ammonia Oxidizer ◽  
Ammonium Transport ◽  
Ammonia Oxidizers ◽  
Batch Cultures ◽  
Ph Values ◽  
Nitrite Production

ABSTRACT Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase

2020 ◽  
Author(s):  
Konstantin Laun ◽  
Iuliia Baranova ◽  
Jifu Duan ◽  
Leonie Kertess ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
H2 Evolution ◽  
Proton Reduction ◽  
Ph Values ◽  
Site Selective ◽  
H2 Oxidation ◽  
Diiron Site

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high oxidation activities, the majority of [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands that facilitate catalysis at low overpotential. Distinct proton transfer pathways connect the active site niche with the solvent, resulting in a non-trivial dependence of hydrogen turnover and bulk pH. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the natural pH dependence of hydrogen turnover as catalyzed by [FeFe]-hydrogenase.<br>

scholarly journals Measurements of nitrite production in and around the primary nitrite maximum in the central California Current

2013 ◽  
Vol 10 (11) ◽  
pp. 7395-7410 ◽  
Author(s):  
A. E. Santoro ◽  
C. M. Sakamoto ◽  
J. M. Smith ◽  
J. N. Plant ◽  
A. L. Gehman ◽  
...  
Keyword(s):  
Ammonia Oxidation ◽  
Heterotrophic Bacteria ◽  
California Current ◽  
Euphotic Zone ◽  
Ammonia Oxidizing Bacteria ◽  
Central California ◽  
Oxidation Rates ◽  
Ammonia Oxidizing Archaea ◽  
Nitrite Production ◽  
Nh3 Oxidation

Abstract. Nitrite (NO2−) is a substrate for both oxidative and reductive microbial metabolism. NO2− accumulates at the base of the euphotic zone in oxygenated, stratified open-ocean water columns, forming a feature known as the primary nitrite maximum (PNM). Potential pathways of NO2− production include the oxidation of ammonia (NH3) by ammonia-oxidizing bacteria and archaea as well as assimilatory nitrate (NO3−) reduction by phytoplankton and heterotrophic bacteria. Measurements of NH3 oxidation and NO3− reduction to NO2− were conducted at two stations in the central California Current in the eastern North Pacific to determine the relative contributions of these processes to NO2− production in the PNM. Sensitive (< 10 nmol L−1), precise measurements of [NH4+] and [NO2−] indicated a persistent NH4+ maximum overlying the PNM at every station, with concentrations as high as 1.5 μmol L−1. Within and just below the PNM, NH3 oxidation was the dominant NO2− producing process, with rates of NH3 oxidation to NO2− of up to 31 nmol L−1 d−1, coinciding with high abundances of ammonia-oxidizing archaea. Though little NO2− production from NO3− was detected, potentially nitrate-reducing phytoplankton (photosynthetic picoeukaryotes, Synechococcus, and Prochlorococcus) were present at the depth of the PNM. Rates of NO2− production from NO3− were highest within the upper mixed layer (4.6 nmol L−1 d−1) but were either below detection limits or 10 times lower than NH3 oxidation rates around the PNM. One-dimensional modeling of water column NO2− production agreed with production determined from 15N bottle incubations within the PNM, but a modeled net biological sink for NO2− just below the PNM was not captured in the incubations. Residence time estimates of NO2− within the PNM ranged from 18 to 470 days at the mesotrophic station and was 40 days at the oligotrophic station. Our results suggest the PNM is a dynamic, rather than relict, feature with a source term dominated by ammonia oxidation.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

2002 ◽  
Vol 283 (5) ◽  
pp. L1117-L1124 ◽  
Author(s):  
Nader G. Abraham ◽  
Shuo Quan ◽  
Paul A. Mieyal ◽  
Liming Yang ◽  
Theresa Burke-Wolin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Fourfold Increase ◽  
Pulmonary Endothelial Cells ◽  
Intracellular Cgmp ◽  
Nitrite Production ◽  
Pulmonary Cells ◽  
Steady State Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3′,5′-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

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