Comprehensive evaluation of primer pairs targeting the ammonia monooxygenase subunit A gene of complete ammonia-oxidizing Nitrospira.

Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.

Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea.

Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms.

Drug discovery-based approach identifies new nitrification inhibitors.

Nitrogen (N) fertilization is crucial to sustain global food security, but fertilizer N production is energy-demanding and subsequent environmental N losses contribute to biodiversity loss and climate change. N losses can be mitigated be interfering with microbial nitrification, and therefore the use of nitrification inhibitors in enhanced efficiency fertilizers (EEFs) is an important N management strategy to increase N use efficiency and reduce N pollution. However, currently applied nitrification inhibitors have limitations and do not target all nitrifying microorganisms. Here, to identify broad-spectrum nitrification inhibitors, we adopted a drug discovery-based approach and screened 45,400 small molecules on different groups of nitrifying microorganisms. Although a high number of potential nitrification inhibitors were identified, none of them targeted all nitrifier groups. Moreover, a high number of new nitrification inhibitors were shown to be highly effective in culture but did not reduce ammonia consumption in soil. One archaea-targeting inhibitor was not only effective in soil, but even reduced - when co-applied with a bacteria-targeting inhibitor - ammonium consumption and greenhouse gas emissions beyond what is achieved with currently applied nitrification inhibitors. This advocates for combining different types of nitrification inhibitors in EEFs to optimize N management practices and make agriculture more sustainable.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria.

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island.

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of "Candidatus Methylacidithermus pantelleriae" PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, "Ca. Methylacidithermus pantelleriae" might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.

Methylacidimicrobium thermophilum AP8, a Novel Methane- and Hydrogen-Oxidizing Bacterium Isolated From Volcanic Soil on Pantelleria Island, Italy.

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60°C), low pH (3-5) and hydrothermal gas emissions mainly composed of carbon dioxide (CO2), methane (CH4), and hydrogen (H2). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60°C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at µmax (0.051 ± 0.001 h-1, doubling time ~14 h) of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3-5 at 50°C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 ± 1 µM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium.