A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria humidicola

A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant–soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO₂⁻ production (r ² = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed ‘biological nitrification inhibition’ (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 μM AT in an assay containing 18.9 mM of NH₄⁺ is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15–25 AT unit g⁻¹ root dry wt day⁻¹). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots (r ² = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO₃⁻ formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures.     

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny,physiology and stable isotope fractionation

Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the North Pacific Ocean that have been maintained in laboratory culture for over 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98–99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH₃) to nitrite (NO₂⁻). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (¹⁵ɛ_NH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal ¹⁵ɛ_NH3 ranged from 13‰ to 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by ¹⁵ɛ_NH3 is similar.     

抑制剂在氨氧化微生物研究中的应用

在氨氧化微生物的相关研究中经常使用各类抑制剂,包括针对硝化作用的抑制剂和针对微生物生长的抑制剂。自发现氨氧化古菌以来,人们在氨氧化细菌抑制剂的基础上重新筛选和使用不同的抑制剂来满足氨氧化微生物研究的需求。抑制剂既可以加速氨氧化古菌的富集,也可以帮助研究者区分古菌与细菌对硝化作用的贡献以及它们自身合成代谢能力的差别。本文综述了各类抑制剂的使用浓度和抑制效果,包括双氰胺(DCD)、3,4-二甲基吡啶磷酸盐(DMPP)、丙烯基硫脲(ATU)等传统抑制剂,乙炔和辛炔等炔烃类抑制剂,一氧化氮清除剂以及抗生素等对氨氧化微生物的活性和生长有特异性或通用抑制能力的抑制剂。通过对氨氧化微生物抑制剂的归纳总结,可为氨氧化微生物研究过程中抑制剂的选择提供参考。     

Inhibition of Ammonia Oxidation in Nitrosomonas europaea by Sulfur Compounds: Thioethers Are Oxidized to Sulfoxides by Ammonia Monooxygenase

Organic sulfur compounds are well-known nitrification inhibitors. The inhibitory effects of dimethylsulfide, dimethyldisulfide, and ethanethiol on ammonia oxidation by Nitrosomonas europaea were examined. Both dimethylsulfide and dimethyldisulfide were weak inhibitors of ammonia oxidation and exhibited inhibitory characteristics typical of substrates for ammonia monooxygenase (AMO). Depletion of dimethylsulfide required O(2) and was prevented with either acetylene or allylthiourea, two inhibitors of AMO. The inhibition of ammonia oxidation by dimethylsulfide was examined in detail. Cell suspensions incubated in the presence of ammonia oxidized dimethylsulfide to dimethyl sulfoxide. Depletion of six other thioethers was also prevented by treating cell suspensions with either allylthiourea or acetylene. The oxidative products of three thioethers were identified as the corresponding sulfoxides. The amount of sulfoxide formed accounted for a majority of the amount of sulfide depleted. By using gas chromatography coupled with mass spectrometry, allylmethylsulfide was shown to be oxidized to allylmethylsulfoxide by N. europaea with the incorporation of a single atom of O derived from O(2) into the sulfide. This result supported our conclusion that a monooxygenase was involved in the oxidation of allylmethylsulfide. The thioethers are concluded to be a new class of substrates for AMO. This is the first report of the oxidation of the sulfur atom by AMO in whole cells of N. europaea. The ability of N. europaea to oxidize dimethylsulfide is not unique among the ammonia-oxidizing bacteria. Nitrosococcus oceanus, a marine nitrifier, was also demonstrated to oxidize dimethylsulfide to dimethyl sulfoxide.     

Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10³ to 10⁴ CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems.     

Factors Limiting Aliphatic Chlorocarbon Degradation by Nitrosomonas europaea: Cometabolic Inactivation of Ammonia Monooxygenase and Substrate Specificity

The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O(2) uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of the enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with [C]TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with C. Treatment of cells with allylthiourea or acetylene prior to incubation with [C]TCE prevented incorporation of C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons could be divided into three classes depending on their biodegradability and inactivating potential: (i) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells; (ii) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.     

Biodegradation of Halogenated Hydrocarbon Fumigants by Nitrifying Bacteria

Three species of nitrifying bacteria were tested for the ability to degrade the halocarbon fumigants methyl bromide, 1,2-dichloropropane, and 1,2-dibromo-3-chloropropane. The soil nitrifiers Nitrosomonas europaea and Nitrosolobus multiformis degraded all three fumigants, while the marine nitrifier Nitrosococcus oceanus degraded only methyl bromide under the conditions tested. Inhibition of biodegradation by allylthiourea and acetylene, specific inhibitors of ammonia monooxygenase, suggests that ammonia monooxygenase is the enzyme which catalyzes fumigant degradation.     

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, ¹⁵N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.     

The effect of methane and methanol on the terrestrial ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’

The ammonia monooxygenase (AMO) is a key enzyme in ammonia-oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper-containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co-oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well-established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (K_i) for methane and methanol were 2.2 and 20 μM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia-oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in ‘Ca. Nitrosocosmicus franklandus C13’ in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one-carbon (C1) compounds in ammonia-oxidizing archaea.     

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

Metabolism of Inorganic N Compounds by Ammonia-Oxidizing Bacteria

ABSTRACT

Ammonia oxidizing bacteria extract energy for growth from the oxidation of ammonia to nitrite. Ammonia monooxygenase, which initiates ammonia oxidation, remains enigmatic given the lack of purified preparations. Genetic and biochemical studies support a model for the enzyme consisting of three subunits and metal centers of copper and iron. Knowledge of hydroxylamine oxidoreductase, which oxidizes hydroxylamine formed by ammonia monooxygenase to nitrite, is informed by a crystal structure and detailed spectroscopic and catalytic studies. Other inorganic nitrogen compounds, including NO, N₂O, NO₂, and N₂ can be consumed and/or produced by ammonia-oxidizing bacteria. NO and N₂O can be produced as byproducts of hydroxylamine oxidation or through nitrite reduction. NO₂ can serve as an alternative oxidant in place of O₂ in some ammonia-oxidizing strains. Our knowledge of the diversity of inorganic N metabolism by ammonia-oxidizing bacteria continues to grow. Nonetheless, many questions remain regarding the enzymes and genes involved in these processes and the role of these pathways in ammonia oxidizers.     

Pacific Northwest Marine Sediments Contain Ammonia-Oxidizing Bacteria in the β Subdivision of the Proteobacteria

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while β-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that γ-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.     

Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O₂, NO₂⁻, and NO₃⁻ profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Diversity, Physiology, and Niche Differentiation of Ammonia-Oxidizing Archaea

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, has been suggested to have been a central part of the global biogeochemical nitrogen cycle since the oxygenation of Earth. The cultivation of several ammonia-oxidizing archaea (AOA) as well as the discovery that archaeal ammonia monooxygenase (amo)-like gene sequences are nearly ubiquitously distributed in the environment and outnumber their bacterial counterparts in many habitats fundamentally revised our understanding of nitrification. Surprising insights into the physiological distinctiveness of AOA are mirrored by the recognition of the phylogenetic uniqueness of these microbes, which fall within a novel archaeal phylum now known as Thaumarchaeota. The relative importance of AOA in nitrification, compared to ammonia-oxidizing bacteria (AOB), is still under debate. This minireview provides a synopsis of our current knowledge of the diversity and physiology of AOA, the factors controlling their ecology, and their role in carbon cycling as well as their potential involvement in the production of the greenhouse gas nitrous oxide. It emphasizes the importance of activity-based analyses in AOA studies and formulates priorities for future research.     

Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.     

Archaea dominate the ammonia-oxidizing community in the rhizosphere of the freshwater macrophyte Littorella uniflora

Archaeal and bacterial ammonia monooxygenase genes (amoA) had similar low relative abundances in freshwater sediment. In the rhizosphere of the submersed macrophyte Littorella uniflora, archaeal amoA was 500- to >8,000-fold enriched compared to bacterial amoA, suggesting that the enhanced nitrification activity observed in the rhizosphere was due to ammonia-oxidizing Archaea.     

Assessment of Changes in Microbial Community Structure during Operation of an Ammonia Biofilter with Molecular Tools

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria.