Wagner, Michael

Summary Marine sponges represent one of the few eukaryotic groups that frequently harbour symbiotic members of the Thaumarchaeota , which are important chemoautotrophic ammonia‐oxidizers in many environments. However, in most studies, direct demonstration of ammonia‐oxidation by these archaea within sponges is lacking, and little is known about sponge‐specific adaptations of ammonia‐oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope‐based functional assays. ‘ Candidatus Nitrosospongia ianthellae’ is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbour nitrite‐oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin‐like proteases, a metalloprotease unique among archaea, as well as a putative branched‐chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge‐associated, but not in free‐living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic‐like proteins, and a DNA‐phosporothioation system, represent important adaptations of AOA to life within these ancient filter‐feeding animals.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO 2 using the Wood–Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N 2 , and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

ABSTRACT Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “ Candidatus Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “ Ca . Nitrotoga” culture was available. Here we obtained the first “ Ca . Nitrotoga” isolate from activated sludge. This organism, “ Candidatus Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “ Ca . Nitrotoga” enrichments, which were described as cold-adapted NOB. “ Ca . Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO 2 − ) and nitrate (up to 25 mM NO 3 − ). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent K m ( K m (app) ) of ~89 µM nitrite and a V max of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “ Ca . Nitrotoga fabula” were reconstructed from the closed genome. “ Ca . Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “ Ca . Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “ Ca . Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “ Ca . Nitrotoga fabula” to survive nitrite depletion and colonize new niches. IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus “ Candidatus Nitrotoga” and performed a detailed physiological and genomic characterization of this organism (“ Candidatus Nitrotoga fabula”). Differences between key phenotypic properties of “ Ca . Nitrotoga fabula” and those of previously enriched “ Ca . Nitrotoga” members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that “ Ca . Nitrotoga” are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in “ Ca . Nitrotoga fabula” expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.

AbstractAmmonia-oxidizing archaea (AOA) within the phylumThaumarchaeaare the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from theNitrosocaldusclade, group 1.1b and group 1.1aThaumarchaeain terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 °C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a novelThaumarchaeonfrom the deep-branchingNitrosocaldaceaefamily of which we have obtained a high (∼85 %) enrichment from biofilm of an Icelandic hot spring (73 °C). This AOA, which we provisionally refer to as “CandidatusNitrosocaldus islandicus”, is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 °C and 70 °C.Ca.N. islandicus encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks anirKgene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent asCa.N. islandicus is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed thatCa.N. islandicus has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase(iorAB)as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes - one of the predominant replicative DNA polymerases in all other ammonia-oxidizingThaumarchaea.Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.

Abstract Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 mm of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca. N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1–2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota, a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ. However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota, suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.

Abstract Ni.tro.so.te.nu.a.ce'ae. N.L. adj nitrosus nitrous; L. adj. tenuis slender; ‐aceae ending to denote a family; N.L. fem. pl. n. Nitrosotenuaceae the Nitrosotenuis family. Candidatus Nitrosotenuaceae: A family of slender nitrite producers Candidatus Nitrosotenuaceae is a family of Thaumarchaeota that consists of a single genus, Ca . Nitrosotenuis, which can be found widely distributed in soils, freshwater, hot springs, the subsurface, and activated sludge. They may be rods or spheres and may or may not have flagella. Similar to other known and described Thaumarchaeota , Ca . Nitrosotenuaceae are aerobic chemolithautotrophs that use energy gained from the oxidation of ammonia to nitrite to fix carbon via modified 3‐hydroxypropionate/4‐hydroxybutyrate carbon fixation pathway. At this time, no pure culture of Ca . Nitrosotenuaceae exists, but some of the six available enrichments with members of the genus are almost pure. It remains to be demonstrated whether the remaining bacterial contaminants provide essential compounds to Ca . Nitrosotenuaceae. Enrichments of Ca . Nitrosotenuaceae are intolerant to high salinity (>0.3%), although they are phylogenetically related to the Group I.1a Thaumarchaeota ( Ca . Nitrosopumilaceae), which includes taxa that are widely distributed in the ocean. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the family Candidatus Nitrosotenuaceae is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Thermoproteati / Thermoproteota / Nitrososphaeria / Nitrosopumilales / Candidatus Nitrosotenuaceae The family Candidatus Nitrosotenuaceae can also be recovered in the Genome Taxonomy Database (GTDB) as f__Nitrosopumilaceae (version v220) ** . GTDB classification: d__Archaea / p__Thermoproteota / c__Nitrososphaeria / o__Nitrososphaerales / f__Nitrosopumilaceae * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract Ni.tro.so.te'nu.is. N.L. adj. nitrosus, full of natron; here intended to mean nitrous; L. masc. adj. tenuis slender; N.L. masc. n. Candidatus Nitrosotenuis a slender nitrite producer. Candidatus Nitrosotenuis: a slender nitrite producer. Candidatus Nitrosotenuis is a genus of Thaumarchaeota that can be found widely distributed in soils, freshwater, hot springs, subsurface, and activated sludge. They may be rods or spheres and may or may not have flagella. Similar to other known and described Thaumarchaeota , Ca . Nitrosotenuis are aerobic chemolithautotrophs that use energy gained from the oxidation of ammonia to nitrite to fix carbon via the modified 3‐hydroxypropionate/4‐hydroxybutyrate carbon fixation pathway. At this time, no pure culture of Ca . Nitrosotenuis exists, but some of the six available enrichments have only a few rare contaminants. It remains to be demonstrated whether the remaining bacterial contaminants provide essential compounds to Ca . Nitrosotenuis. Enrichments of Ca . Nitrosotenuis are intolerant to high salinity (>0.3%), although they are phylogenetically placed within the group I.1a Thaumarchaeota ( Ca . Nitrosopumilales), which includes taxa that are widely distributed in the ocean. DNA G + C content (mol%) : 41.0–42.2. Type species : Candidatus Nitrosotenuis uzonensis . Lebedeva, Hatzenpichler, Pelletier, Schuster, Hauzmayer, Bulaev, Grigor'eva, Galushko, Schmid, Palatinszky, Le Paslier, Daims and Wagner 2013, 8. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Nitrosotenuis is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Thermoproteati / Thermoproteota / Nitrososphaeria / Nitrosopumilales / Candidatus Nitrosotenuaceae / Candidatus Nitrosotenuis The genus Candidatus Nitrosotenuis can also be recovered in the Genome Taxonomy Database (GTDB) as g__Nitrosotenuis (version v220) ** . GTDB classification: d__Archaea / p__Thermoproteota / c__Nitrososphaeria / o__Nitrososphaerales / f__Nitrosopumilaceae / g__Nitrosotenuis * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract The candidate archaeal phylum ‘Aigarchaeota’ contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous ‘streamer’ community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus ‘Calditenuis aerorheumensis’ for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.