Zhen-Hao Luo

As key drivers of nitrification, ammonia-oxidizing archaea (AOA) play a central role in the global nitrogen cycle and contribute significantly to the emissions of the potent greenhouse gas nitrous oxide (N ₂ O). However, the ecological implications of AOA growth as biofilms, remain poorly understood. Since nitrite production can be used to follow cellular activities directly we were able to compare biofilms with planktonic cells of the terrestrial model AOA Nitrososphaera viennensis at ecologically and agriculturally relevant conditions. Biofilms were more resistant across nearly all tested conditions and remained active at lower temperatures, acidic pH, and high ammonium concentrations. Collectively, activities in biofilm help reconcile discrepancies between earlier laboratory and environmental observations of soil AOA. Additionally, biofilms showed a high general resilience and lowered sensitivities to nitrification inhibitors. Although in situ biofilms grown in microrespiratory chambers exhibited activity and ammonia affinity similar to planktonic cells, biofilm cultures produced only half as much N₂O. The enhanced fitness of biofilms across all tested conditions vastly expands the potential ecophysiological niche of AOA and supports the hypothesis that biofilm growth represents the in situ phenotype of AOA in soil environments.

Asgard archaea are considered the closest prokaryotic relatives of eukaryotes and having a crucial role in eukaryogenesis. Only few organisms have been cultivated from this group and their viruses were previously described only through metagenomic reconstructions. Here, we report the first successful cultivation of an Asgard archaeal virus infecting a novel strain of Ca. Lokiarchaeum ossiferum B36. The 16 kbp integrated provirus is capable to excise and replicate independently leading to the formation of virus particles. Network analysis of shared protein clusters with other archaeal viruses places it in a new family, Fylgjaviridae . The host encodes a distinctive repertoire of antiviral defense systems, including Septu, Wadjet, and type II CBASS system, all different from the defense systems of the related strain Ca. L. ossiferum B35. Our cultures provide the first opportunity to study interactions of a virus–host system in Asgards and hold significant potential for developing genetic tools.

The CPR and DPANN superphyla are globally distributed in anoxic habitats including extreme environments. However, the biogeography and potential ecological functions of their viruses remain unexplored. Here, we recover diverse CPR/DPANN metagenomic viral genomes from 90 acid mine drainage (AMD) sediments sampled across southeast China. Our data reveal deterministic processes as the primary driver of virome assembly shaping the distinct distribution patterns of CPR and DPANN viruses. While lifestyle prediction shows higher lytic virus diversity associated with DPANN, both CPR/DPANN viruses likely use the Piggyback-the-winner (PtW) strategy to co-exist with hosts in AMD sediments, with CPR viromes exhibiting increased lysis in low host-density regimes under intensive acidity/salinity conditions. A subsequent metatranscriptomic analysis uncovers diverse functional genes encoded by CPR and DPANN viruses actively expressed in situ, potentially supplementing host metabolisms yet diverging in replication, transcription, and translation-related functions. Furthermore, partial correlation network analysis suggests that putative symbiotic hosts of the CPR/DPANN may confer protection against viral infection through enhanced antiviral defense. Our results highlight the complex interplays between viruses, DPANN and CPR organisms, and their symbiotic hosts.

The ubiquitously distributed ammonia-oxidizing archaea generate energy from ammonia and build cell mass from inorganic carbon sources, thereby contributing to both the global nitrogen and carbon cycles. However, little is known about the regulation of their predicted core carbon metabolism. A thermodynamic model for Nitrososphaera viennensis was developed to estimate the consumption of inorganic carbon in relation to ammonia consumed for energy and was tested experimentally by growing cells in carbon-limited and excess conditions. A combined proteomic and metabolomic approach to the experimental conditions revealed distinct metabolic adaptation depending on the amount of carbon supplied, either in a catalase or pyruvate background as a reactive oxygen species scavenger. Integration of protein and metabolite dynamics revealed a cellular strategy under carbon limitation to maintain a pool of amino acids and an upregulation of proteins necessary for translation initiation to stay primed for protein synthesis. The combination of modeling and functional genomics fills gaps in the understanding of the central metabolism and its regulation in a chemolithoautotrophic, ammonia-oxidizing archaeon, even in the absence of available genetic tools. IMPORTANCE: Little is known about the regulation of carbon metabolism within ammonia-oxidizing archaea (AOA), a widespread clade that plays a critical role in the global nitrogen cycle while also fixing inorganic carbon. To address this missing knowledge, the soil AOA Nitrososphaera viennensis was subjected to various levels of inorganic carbon and analyzed via a systems biology approach to better understand how its core metabolism is regulated. The results demonstrate a strong dependence on the carbon fixation cycle and highlight key connection points between the core metabolic pathways. The analysis additionally revealed tight control on translational processes and elucidated unique cellular responses when the organism was exposed to either exogenous catalase or pyruvate to relieve oxidative stress from reactive oxygen species. The presented data highlight metabolic responses of N. viennensis and provide a better understanding of how the organism, and likely other AOA, respond to various environmental conditions.

Recent genomic surveys have uncovered candidate phyla radiation (CPR) bacteria and DPANN archaea as major microbial dark matter lineages in various anoxic habitats. Despite their extraordinary diversity, the biogeographic patterns and ecological implications of these ultra-small and putatively symbiotic microorganisms have remained elusive. Here, we performed metagenomic sequencing on 90 geochemically diverse acid mine drainage sediments sampled across southeast China and recovered 282 CPR and 189 DPANN nonredundant metagenome-assembled genomes, which collectively account for up to 28.6% and 31.2% of the indigenous prokaryotic communities, respectively. We found that, remarkably, geographic distance represents the primary factor driving the large-scale ecological distribution of both CPR and DPANN organisms, followed by pH and Fe. Although both groups might be capable of iron reduction through a flavin-based extracellular electron transfer mechanism, significant differences are found in their metabolic capabilities (with complex carbon degradation and chitin degradation being more prevalent in CPR whereas fermentation and acetate production being enriched in DPANN), indicating potential niche differentiation. Predicted hosts are mainly Acidobacteriota , Bacteroidota , and Proteobacteria for CPR and Thermoplasmatota for DPANN, and extensive, unbalanced metabolic exchanges between these symbionts and putative hosts are displayed. Together, our results provide initial insights into the complex interplays between the two lineages and their physicochemical environments and host populations at a large geographic scale. IMPORTANCE: Candidate phyla radiation (CPR) bacteria and DPANN archaea constitute a significant fraction of Earth’s prokaryotic diversity. Despite their ubiquity and abundance, especially in anoxic habitats, we know little about the community patterns and ecological drivers of these ultra-small, putatively episymbiotic microorganisms across geographic ranges. This study is facilitated by a large collection of CPR and DPANN metagenome-assembled genomes recovered from the metagenomes of 90 sediments sampled from geochemically diverse acid mine drainage (AMD) environments across southeast China. Our comprehensive analyses have allowed first insights into the biogeographic patterns and functional differentiation of these major enigmatic prokaryotic groups in the AMD model system.

Although ammonia-oxidizing archaea (AOA) are globally distributed in nature, growth in biofilms has been relatively little explored. Here, we investigated six representatives of three different terrestrial and marine clades of AOA in a longitudinal and quantitative study for their ability to form biofilm, and studied gene expression patterns of three representatives. Although all strains grew on a solid surface, soil strains of the genera Nitrosocosmicus and Nitrososphaera exhibited the highest capacity for biofilm formation. Based on microscopic and gene expression data, two different colonization strategies could be distinguished. S-layer containing AOA (from both soil and marine habitats) initialized attachment as single cells, subsequently forming denser layers, whereas the S-layer free species of the Nitrosocosmicus clade attached as suspended aggregates to the surface and henceforth showed fastest establishment of biofilm. Transcription profiles were significantly different between planktonic and biofilm growth in all strains, and revealed individual transcriptomic responses, albeit fulfilling shared functions. In particular, the strong expression of different types of multicopper oxidases was observed in all strains suggesting modifications of their cell coats. S-layer carrying AOA each additionally expressed a set of adhesion proteins supporting attachment. Detoxification of nitrous compounds, copper acquisition as well as the expression of transcription factor B were also shared responses among biofilm producing strains. However, the majority of differentially expressed protein families was distinct among the three strains, illustrating that individual solutions have evolved for the shared growth mode of biofilm formation in AOA, probably driven by the different ecological niches.

Although widespread in nature, growth in biofilms has been relatively little explored in the globally distributed ammonia oxidizing archaea (AOA). Here we investigated six representatives of three different terrestrial and marine clades of AOA in a longitudinal and quantitative study for their ability to form biofilm and studied gene expression patterns of three representatives. While all strains grew on a solid surface, soil strains exhibited the highest capacity for biofilm formation. Based on microscopic and gene expression data, two different colonization strategies could be distinguished. S-layer containing AOA (from both soil and marine habitats) initialized attachment as single cells and subsequently formed denser layers and three-dimensional structures, while the S-layer free species of the Nitrosocosmicus clade attached as suspended aggregates to the surface and henceforth showed fastest establishment of biofilm. Transcription profiles were significantly different between planktonic and biofilm growth in all strains and revealed individual reactions, often fulfilling shared functions. In particular the strong expression of different types of multicopper oxidases was observed in all strains indicating modifications of their cell coats. S-layer carrying AOA each additionally expressed a set of adhesion proteins supporting attachment. Detoxification of nitrous compounds, copper acquisition as well as the expression of transcription factor B were also shared reactions among biofilm producing strains. However, the majority of differentially expressed protein families was distinct among the three strains illustrating that individual solutions have evolved for the shared growth mode of biofilm formation in AOA, probably driven by the different ecological niches.