Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe

The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene (amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

The model structure of the copper-dependent ammonia monooxygenase

Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract

Effects of Zeolite and Biochar Addition on Ammonia-Oxidizing Bacteria and Ammonia-Oxidizing Archaea Communities during Agricultural Waste Composting

The effects of zeolite and biochar addition on ammonia-oxidizing bacteria (AOB) and archaea (AOA) communities during agricultural waste composting were determined in this study. Four treatments were conducted as follows: Treatment A as the control with no additive, Treatment B with 5% of zeolite, Treatment C with 5% of biochar, and Treatment D with 5% of zeolite and 5% biochar, respectively. The AOB and AOA amoA gene abundance as well as the ammonia monooxygenase (AMO) activity were estimated by quantitative PCR and enzyme-linked immunosorbent assay, respectively. The relationship between gene abundance and AMO enzyme activity was determined by regression analysis. Results indicated that the AOB was more abundant than that of AOA throughout the composting process. Addition of biochar and its integrated application with zeolite promoted the AOB community abundance and AMO enzyme activity. Significant positive relationships were obtained between AMO enzyme activity and AOB community abundance (r2 = 0.792; P < 0.01) and AOA community abundance (r2 = 0.772; P < 0.01), indicating that both bacteria and archaea played significant roles in microbial ammonia oxidation during composting. Using biochar and zeolite might promote the nitrification activity by altering the sample properties during agricultural waste composting.

MetaFunPrimer: primer design for targeting genes observed in metagenomes

ABSTRACTHigh throughput primer design is needed to simultaneously design primers for multiple genes of interest, such as a group of functional genes. We have developed MetaFunPrimer, a bioinformatic pipeline to design primer targets for genes of interests, with a prioritization based on ranking the presence of gene targets in references, such as metagenomes. MetaFunPrimer takes inputs of protein and nucleotide sequences for gene targets of interest accompanied by a set of reference metagenomes or genomes for determining genes of interest. Its output is a set of primers that may be used to amplify genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 HT-qPCR primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 soil metagenomes. We demonstrate that these primers can significantly improve targeting of amoA-AOB genes in soil metagenomes compared to previously published primers.IMPORTANCEAmplification-based gene characterization allows for sensitive and specific quantification of functional genes. Often, there is a large diversity of genes represented for a function of interest, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high throughput primer design for functional genes of interest and also allows for ranking gene targets by their presence and abundance in environmental datasets. This tool enables high throughput qPCR approaches for characterizing functional genes.

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

ABSTRACT Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, “Candidatus Nitrosocosmicus franklandus” C13 and “Candidatus Nitrosotalea sinensis” Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from “Ca. Nitrosocosmicus franklandus” and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits “Ca. Nitrosocosmicus franklandus” and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from “Ca. Nitrosocosmicus franklandus,” and it does not compete with NH3 for binding at the active site. IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Effects of urease and nitrification inhibitors on soil N, nitrifier abundance and activity in a sandy loam soil

Inhibitors of urease and ammonia monooxygenase can limit the rate of conversion of urea to ammonia and ammonia to nitrate, respectively, potentially improving N fertilizer use efficiency and reducing gaseous losses. Winter wheat grown on a sandy soil in the UK was treated with urea fertilizer with the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT), the nitrification inhibitor dicyandiamide (DCD) or a combination of both. The effects on soil microbial community diversity, the abundance of genes involved in nitrification and crop yields and net N recovery were compared. The only significant effect on N-cycle genes was a transient reduction in bacterial ammonia monooxygenase abundance following DCD application. However, overall crop yields and net N recovery were significantly lower in the urea treatments compared with an equivalent application of ammonium nitrate fertilizer, and significantly less for urea with DCD than the other urea treatments.