Phylogenetics and environmental distribution of nitric oxide-forming nitrite reductases reveal their distinct functional and ecological roles.

The two evolutionarily unrelated nitric oxide-producing nitrite reductases, NirK and NirS, are best known for their redundant role in denitrification. They are also often found in organisms that do not perform denitrification. To assess the functional roles of the two enzymes and to address the sequence and structural variation within each, we reconstructed robust phylogenies of both proteins with sequences recovered from 6973 isolate and metagenome-assembled genomes and identified 32 well-supported clades of structurally distinct protein lineages. We then inferred the potential niche of each clade by considering other functional genes of the organisms carrying them as well as the relative abundances of each nir gene in 4082 environmental metagenomes across diverse aquatic, terrestrial, host-associated, and engineered biomes. We demonstrate that Nir phylogenies recapitulate ecology distinctly from the corresponding organismal phylogeny. While some clades of the nitrite reductase were equally prevalent across biomes, others had more restricted ranges. Nitrifiers make up a sizeable proportion of the nitrite-reducing community, especially for NirK in marine waters and dry soils. Furthermore, the two reductases showed distinct associations with genes involved in oxidizing and reducing other compounds, indicating that the NirS and NirK activities may be linked to different elemental cycles. Accordingly, the relative abundance and diversity of NirS versus NirK vary between biomes. Our results show the divergent ecological roles NirK and NirS-encoding organisms may play in the environment and provide a phylogenetic framework to distinguish the traits associated with organisms encoding the different lineages of nitrite reductases.

Diversity and ecology of NrfA-dependent ammonifying microorganisms.

Nitrate ammonifiers are a taxonomically diverse group of microorganisms that reduce nitrate to ammonium, which is released, and thereby contribute to the retention of nitrogen in ecosystems. Despite their importance for understanding the fate of nitrate, they remain a largely overlooked group in the nitrogen cycle. Here, we present the latest advances on free-living microorganisms using NrfA to reduce nitrite during ammonification. We describe their diversity and ecology in terrestrial and aquatic environments, as well as the environmental factors influencing the competition for nitrate with denitrifiers that reduce nitrate to gaseous nitrogen species, including the greenhouse gas nitrous oxide (N2O). We further review the capacity of ammonifiers for other redox reactions, showing that they likely play multiple roles in the cycling of elements.

Phyloecology of nitrate ammonifiers and their importance relative to denitrifiers in global terrestrial biomes.

Nitrate ammonification is important for soil nitrogen retention. However, the ecology of ammonifiers and their prevalence compared with denitrifiers, being competitors for nitrate, are overlooked. Here, we screen 1 million genomes for nrfA and onr, encoding ammonifier nitrite reductases. About 40% of ammonifier assemblies carry at least one denitrification gene and show higher potential for nitrous oxide production than consumption. We then use a phylogeny-based approach to recruit gene fragments of nrfA, onr and denitrification nitrite reductase genes (nirK, nirS) in 1861 global terrestrial metagenomes. nrfA outnumbers the nearly negligible onr counts in all biomes, but denitrification genes dominate, except in tundra. Random forest modelling teases apart the influence of the soil C/N on nrfA-ammonifier vs denitrifier abundance, showing an effect of nitrate rather than carbon content. This study demonstrates the multiple roles nitrate ammonifiers play in nitrogen cycling and identifies factors ultimately controlling the fate of soil nitrate.

Distribution and Environmental Drivers of Fungal Denitrifiers in Global Soils.

The microbial process of denitrification is the primary source of the greenhouse gas nitrous oxide (N2O) from terrestrial ecosystems. Fungal denitrifiers, unlike many bacteria, lack the N2O reductase, and thereby are sources of N2O. Still, their diversity, global distribution, and environmental determinants, as well as their relative importance, compared to bacterial and archaeal denitrifiers, remain unresolved. Employing a phylogenetically informed approach to analyze 1,980 global soil and rhizosphere metagenomes for the denitrification marker gene nirK, which codes for the copper dependent nitrite reductase in denitrification, we show that fungal denitrifiers are sparse, yet cosmopolitan and that they are dominated by saprotrophs and pathogens. Few showed biome-specific distribution patterns, although members of the Fusarium oxysporum species complex, which are known to produce substantial amounts of N2O, were proportionally more abundant and diverse in the rhizosphere than in other biomes. Fungal denitrifiers were most frequently detected in croplands, but they were most abundant in forest soils when normalized to metagenome size. Nevertheless, the overwhelming dominance of bacterial and archaeal denitrifiers suggests a much lower fungal contribution to N2O emissions than was previously estimated. In relative terms, they could play a role in soils that are characterized by a high carbon to nitrogen ratio and a low pH, especially in the tundra as well as in boreal and temperate coniferous forests. Because global warming predicts the proliferation of fungal pathogens, the prevalence of potential plant pathogens among fungal denitrifiers and the cosmopolitan distribution of these organisms suggest that fungal denitrifier abundance may increase in terrestrial ecosystems. IMPORTANCE Fungal denitrifiers, in contrast to their bacterial counterparts, are a poorly studied functional group within the nitrogen cycle, even though they produce the greenhouse gas N2O. To curb soil N2O emissions, a better understanding of their ecology and distribution in soils from different ecosystems is needed. Here, we probed a massive amount of DNA sequences and corresponding soil data from a large number of samples that represented the major soil environments for a broad understanding of fungal denitrifier diversity at the global scale. We show that fungal denitrifiers are predominantly cosmopolitan saprotrophs and opportunistic pathogens. Fungal denitrifiers constituted, on average, 1% of the total denitrifier community. This suggests that earlier estimations of fungal denitrifier abundance, and, thereby, it is also likely that the contributions of fungal denitrifiers to N2O emissions have been overestimated. Nevertheless, with many fungal denitrifiers being plant pathogens, they could become increasingly relevant, as soilborne pathogenic fungi are predicted to increase with ongoing climate change.

The impact of agricultural management on soil aggregation and carbon storage is regulated by climatic thresholds across a 3000 km European gradient.

Organic carbon and aggregate stability are key features of soil quality and are important to consider when evaluating the potential of agricultural soils as carbon sinks. However, we lack a comprehensive understanding of how soil organic carbon (SOC) and aggregate stability respond to agricultural management across wide environmental gradients. Here, we assessed the impact of climatic factors, soil properties and agricultural management (including land use, crop cover, crop diversity, organic fertilization, and management intensity) on SOC and the mean weight diameter of soil aggregates, commonly used as an indicator for soil aggregate stability, across a 3000 km European gradient. Soil aggregate stability (-56%) and SOC stocks (-35%) in the topsoil (20 cm) were lower in croplands compared with neighboring grassland sites (uncropped sites with perennial vegetation and little or no external inputs). Land use and aridity were strong drivers of soil aggregation explaining 33% and 20% of the variation, respectively. SOC stocks were best explained by calcium content (20% of explained variation) followed by aridity (15%) and mean annual temperature (10%). We also found a threshold-like pattern for SOC stocks and aggregate stability in response to aridity, with lower values at sites with higher aridity. The impact of crop management on aggregate stability and SOC stocks appeared to be regulated by these thresholds, with more pronounced positive effects of crop diversity and more severe negative effects of crop management intensity in nondryland compared with dryland regions. We link the higher sensitivity of SOC stocks and aggregate stability in nondryland regions to a higher climatic potential for aggregate-mediated SOC stabilization. The presented findings are relevant for improving predictions of management effects on soil structure and C storage and highlight the need for site-specific agri-environmental policies to improve soil quality and C sequestration.

Agricultural management and pesticide use reduce the functioning of beneficial plant symbionts.

Phosphorus (P) acquisition is key for plant growth. Arbuscular mycorrhizal fungi (AMF) help plants acquire P from soil. Understanding which factors drive AMF-supported nutrient uptake is essential to develop more sustainable agroecosystems. Here we collected soils from 150 cereal fields and 60 non-cropped grassland sites across a 3,000 km trans-European gradient. In a greenhouse experiment, we tested the ability of AMF in these soils to forage for the radioisotope 33P from a hyphal compartment. AMF communities in grassland soils were much more efficient in acquiring 33P and transferred 64% more 33P to plants compared with AMF in cropland soils. Fungicide application best explained hyphal 33P transfer in cropland soils. The use of fungicides and subsequent decline in AMF richness in croplands reduced 33P uptake by 43%. Our results suggest that land-use intensity and fungicide use are major deterrents to the functioning and natural nutrient uptake capacity of AMF in agroecosystems.

Land-use intensification differentially affects bacterial, fungal and protist communities and decreases microbiome network complexity.

BACKGROUND: Soil microbial communities are major drivers of cycling of soil nutrients that sustain plant growth and productivity. Yet, a holistic understanding of the impact of land-use intensification on the soil microbiome is still poorly understood. Here, we used a field experiment to investigate the long-term consequences of changes in land-use intensity based on cropping frequency (continuous cropping, alternating cropping with a temporary grassland, perennial grassland) on bacterial, protist and fungal communities as well as on their co-occurrence networks. RESULTS: We showed that land use has a major impact on the structure and composition of bacterial, protist and fungal communities. Grassland and arable cropping differed markedly with many taxa differentiating between both land use types. The smallest differences in the microbiome were observed between temporary grassland and continuous cropping, which suggests lasting effects of the cropping system preceding the temporary grasslands. Land-use intensity also affected the bacterial co-occurrence networks with increased complexity in the perennial grassland comparing to the other land-use systems. Similarly, co-occurrence networks within microbial groups showed a higher connectivity in the perennial grasslands. Protists, particularly Rhizaria, dominated in soil microbial associations, as they showed a higher number of connections than bacteria and fungi in all land uses. CONCLUSIONS: Our findings provide evidence of legacy effects of prior land use on the composition of the soil microbiome. Whatever the land use, network analyses highlighted the importance of protists as a key element of the soil microbiome that should be considered in future work. Altogether, this work provides a holistic perspective of the differential responses of various microbial groups and of their associations to agricultural intensification.

Diversity of archaea and niche preferences among putative ammonia-oxidizing Nitrososphaeria dominating across European arable soils.

Archaeal communities in arable soils are dominated by Nitrososphaeria, a class within Thaumarchaeota comprising all known ammonia-oxidizing archaea (AOA). AOA are key players in the nitrogen cycle and defining their niche specialization can help predicting effects of environmental change on these communities. However, hierarchical effects of environmental filters on AOA and the delineation of niche preferences of nitrososphaerial lineages remain poorly understood. We used phylogenetic information at fine scale and machine learning approaches to identify climatic, edaphic and geomorphological drivers of Nitrososphaeria and other archaea along a 3000 km European gradient. Only limited insights into the ecology of the low-abundant archaeal classes could be inferred, but our analyses underlined the multifactorial nature of niche differentiation within Nitrososphaeria. Mean annual temperature, C:N ratio and pH were the best predictors of their diversity, evenness and distribution. Thresholds in the predictions could be defined for C:N ratio and cation exchange capacity. Furthermore, multiple, independent and recent specializations to soil pH were detected in the Nitrososphaeria phylogeny. The coexistence of widespread ecophysiological differences between closely related soil Nitrososphaeria highlights that their ecology is best studied at fine phylogenetic scale.

Crop cover is more important than rotational diversity for soil multifunctionality and cereal yields in European cropping systems.

In natural ecosystems, positive effects of plant diversity on ecosystem functioning have been widely observed, yet whether this is true in cropping systems remains unclear. Here we assessed the impact of crop diversification on soil microbial diversity, soil multifunctionality (SMF) and crop yields in 155 cereal fields across a 3,000 km north-south European gradient. Overall, crop diversity showed a relatively minor effect on soil microbial diversity, SMF and yields. In contrast, the proportion of time with crop cover (including cash crops, cover crops or forage leys) during the past ten-year crop rotation had a much stronger impact. This suggests that increasing crop cover can enhance both yields and soil functioning, while also providing habitat for soil microorganisms. We found that SMF did not positively contribute to crop yields, highlighting that care must be taken to balance the provision of food with environmentally beneficial functions and services, since they do not always go hand in hand.

Core microbial communities of lacustrine microbialites sampled along an alkalinity gradient.

Microbialites are usually carbonate-rich sedimentary rocks formed by the interplay of phylogenetically and metabolically complex microbial communities with their physicochemical environment. Yet, the biotic and abiotic determinants of microbialite formation remain poorly constrained. Here, we analysed the structure of prokaryotic and eukaryotic communities associated with microbialites occurring in several crater lakes of the Trans-Mexican volcanic belt along an alkalinity gradient. Microbialite size and community structure correlated with lake physicochemical parameters, notably alkalinity. Although microbial community composition varied across lake microbialites, major taxa-associated functions appeared quite stable with both, oxygenic and anoxygenic photosynthesis and, to less extent, sulphate reduction, as major putative carbonatogenic processes. Despite interlake microbialite community differences, we identified a microbial core of 247 operational taxonomic units conserved across lake microbialites, suggesting a prominent ecological role in microbialite formation. This core mostly encompassed Cyanobacteria and their typical associated taxa (Bacteroidetes, Planctomycetes) and diverse anoxygenic photosynthetic bacteria, notably Chloroflexi, Alphaproteobacteria (Rhodobacteriales, Rhodospirilalles), Gammaproteobacteria (Chromatiaceae) and minor proportions of Chlorobi. The conserved core represented up to 40% (relative abundance) of the total community in lakes Alchichica and Atexcac, displaying the highest alkalinities and the most conspicuous microbialites. Core microbialite communities associated with carbonatogenesis might be relevant for inorganic carbon sequestration purposes.

A Novel Microbialite-Associated Phototrophic Chloroflexi Lineage Exhibiting a Quasi-Clonal Pattern along Depth.

Chloroflexales (Chloroflexi) are typical members of the anoxygenic photosynthesizing component of microbial mats and have mostly been characterized from communities associated to hot springs. Here, we report the assembly of five metagenome-assembled genomes (MAGs) of a novel lineage of Chloroflexales found in mesophilic lithifying microbial mats (microbialites) in Lake Alchichica (Mexico). Genomic and phylogenetic analyses revealed that the bins shared 92% of their genes, and these genes were nearly identical despite being assembled from samples collected along a depth gradient (1-15 m depth). We tentatively name this lineage Candidatus Lithoflexus mexicanus. Metabolic predictions based on the MAGs suggest that these chlorosome-lacking mixotrophs share features in central carbon metabolism, electron transport, and adaptations to life under oxic and anoxic conditions, with members of two related lineages, Chloroflexineae and Roseiflexineae. Contrasting with the other diverse microbialite community members, which display much lower genomic conservation along the depth gradient, Ca. L. mexicanus MAGs exhibit remarkable similarity. This might reflect a particular flexibility to acclimate to varying light conditions with depth or the capacity to occupy a very specific spatial ecological niche in microbialites from different depths. Alternatively, Ca. L. mexicanus may also have the ability to modulate its gene expression as a function of the local environmental conditions during diel cycles in microbialites along the depth gradient.

Functional shifts in microbial mats recapitulate early Earth metabolic transitions.

Phototrophic microbial mats dominated terrestrial ecosystems for billions of years, largely causing, through cyanobacterial oxygenic photosynthesis, but also undergoing, the Great Oxidation Event approximately 2.5 billion years ago. Taking a space-for-time approach based on the universality of core metabolic pathways expressed at ecosystem level, we studied gene content and co-occurrence networks in high-diversity metagenomes from spatially close microbial mats along a steep redox gradient. The observed functional shifts suggest that anoxygenic photosynthesis was present but not predominant under early Precambrian conditions, being accompanied by other autotrophic processes. Our data also suggest that, in contrast to general assumptions, anoxygenic photosynthesis largely expanded in parallel with the subsequent evolution of oxygenic photosynthesis and aerobic respiration. Finally, our observations might represent space-for-time evidence that the Wood-Ljungdahl carbon fixation pathway dominated phototrophic mats in early ecosystems, whereas the Calvin cycle probably evolved from pre-existing variants before becoming the dominant contemporary form of carbon fixation.

Unveiling microbial interactions in stratified mat communities from a warm saline shallow pond.

Modern phototrophic microbial mats are complex communities often used as analogs of major Precambrian ecosystems. Characterizing biotic, notably metabolic, interactions among different microbial mat members is essential to gain insights into the ecology and biogeochemistry of these systems. We applied 16S/18S rRNA metabarcoding approaches to characterize the structure of archaea, bacteria and protist communities from microbial mats collected along strong physicochemical (oxygen, salinity, temperature, depth) gradients in a shallow pond at the salar de Llamara (Chile). All mats were highly diverse, including members of virtually all known high-rank eukaryotic and prokaryotic taxa but also many novel lineages. Bacterial candidate divisions accounted for almost 50% of sequences in deeper mats, while Archaea represented up to 40% of sequences in some mat layers. Molecular phylogenetic analyses revealed six novel deeply divergent archaeal groups, along abundant and diverse Pacearchaeota and Woesearchaeota. Multivariate statistical analyses showed that local environmental conditions strongly influenced community composition. Co-occurrence network structure was markedly different between surface mats located in the oxygenated zone and mats located in transition and anoxic water layers. We identified potential biotic interactions between various high- and low-rank taxa. Notably, a strong positive correlation was observed between Lokiarchaeota and the poorly known candidate bacterial division TA06.

Comparative metagenomics unveils functions and genome features of microbialite-associated communities along a depth gradient.

Modern microbialites are often used as analogs of Precambrian stromatolites; therefore, studying the metabolic interplay within their associated microbial communities can help formulating hypotheses on their formation and long-term preservation within the fossil record. We performed a comparative metagenomic analysis of microbialite samples collected at two sites and along a depth gradient in Lake Alchichica (Mexico). The community structure inferred from single-copy gene family identification and long-contig (>10 kb) assignation, consistently with previous rRNA gene surveys, showed a wide prokaryotic diversity dominated by Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria, and Bacteroidetes, while eukaryotes were largely dominated by green algae or diatoms. Functional analyses based on RefSeq, COG and SEED assignations revealed the importance of housekeeping functions, with an overrepresentation of genes involved in carbohydrate metabolism, as compared with other metabolic capacities. The search for genes diagnostic of specific metabolic functions revealed the important involvement of Alphaproteobacteria in anoxygenic photosynthesis and sulfide oxidation, and Cyanobacteria in oxygenic photosynthesis and nitrogen fixation. Surprisingly, sulfate reduction appeared negligible. Comparative analyses suggested functional similarities among various microbial mat and microbialite metagenomes as compared with soil or oceans, but showed differences in microbial processes among microbialite types linked to local environmental conditions.

Genomes of Abundant and Widespread Viruses from the Deep Ocean.

UNLABELLED: The deep sea is a massive, largely oligotrophic ecosystem, stretched over nearly 65% of the planet's surface. Deep-sea planktonic communities are almost completely dependent upon organic carbon sinking from the productive surface, forming a vital component of global biogeochemical cycles. However, despite their importance, viruses from the deep ocean remain largely unknown. Here, we describe the first complete genomes of deep-sea viruses assembled from metagenomic fosmid libraries. "Candidatus Pelagibacter" (SAR11) phage HTVC010P and Puniceispirillum phage HMO-2011 are considered the most abundant cultured marine viruses known to date. Remarkably, some of the viruses described here recruited as many reads from deep waters as these viruses do in the photic zone, and, considering the gigantic scale of the bathypelagic habitat, these genomes provide information about what could be some of the most abundant viruses in the world at large. Their role in the viral shunt in the global ocean could be very significant. Despite the challenges encountered in inferring the identity of their hosts, we identified one virus predicted to infect members of the globally distributed SAR11 cluster. We also identified a number of putative proviruses from diverse taxa, including deltaproteobacteria, bacteroidetes, SAR11, and gammaproteobacteria. Moreover, our findings also indicate that lysogeny is the preferred mode of existence for deep-sea viruses inhabiting an energy-limited environment, in sharp contrast to the predominantly lytic lifestyle of their photic-zone counterparts. Some of the viruses show a widespread distribution, supporting the tenet "everything is everywhere" for the deep-ocean virome. IMPORTANCE: The deep sea is among the largest known habitats and a critical cog in biogeochemical cycling but remains underexplored in its microbiology. Even more than is the case for its prokaryotic community, our knowledge of its viral component has remained limited by the paucity of information provided by studies dependent upon short sequence fragments. In this work, we attempt to fill this existing gap by using a combination of classical fosmid libraries with next-generation sequencing and assembly to recover long viral genomic fragments. We have sequenced ca. 6,000 fosmids from two metagenomics libraries made from prokaryotic biomass from the deep Mediterranean Sea and recovered twenty-eight complete viral genomes, all of them novel and quite distinct from all previously described viral genomes. They are preferentially found in deeper waters and are widely distributed all over the oceans. To our knowledge, this is the first report on complete and cosmopolitan viral genomes from the bathypelagic habitat.

Metagenome-based diversity analyses suggest a significant contribution of non-cyanobacterial lineages to carbonate precipitation in modern microbialites.

Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms (e.g., anoxygenic photosynthesis, sulfate reduction), may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient (1, 5, 10, and 15 m depth). The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.