Diversity, Physiology, and Niche Differentiation of Ammonia-Oxidizing Archaea

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, has been suggested to have been a central part of the global biogeochemical nitrogen cycle since the oxygenation of Earth. The cultivation of several ammonia-oxidizing archaea (AOA) as well as the discovery that archaeal ammonia monooxygenase (amo)-like gene sequences are nearly ubiquitously distributed in the environment and outnumber their bacterial counterparts in many habitats fundamentally revised our understanding of nitrification. Surprising insights into the physiological distinctiveness of AOA are mirrored by the recognition of the phylogenetic uniqueness of these microbes, which fall within a novel archaeal phylum now known as Thaumarchaeota. The relative importance of AOA in nitrification, compared to ammonia-oxidizing bacteria (AOB), is still under debate. This minireview provides a synopsis of our current knowledge of the diversity and physiology of AOA, the factors controlling their ecology, and their role in carbon cycling as well as their potential involvement in the production of the greenhouse gas nitrous oxide. It emphasizes the importance of activity-based analyses in AOA studies and formulates priorities for future research

Identification and characterization of novel ammonia-oxidizing archaea and bacteria

Nitrifikation, die biologische aerobe Oxidation von Ammoniak zu Nitrat via Nitrit, ist ein essentieller Schritt des biogeochemischen Stickstoff-Zyklus. Die Entdeckung dass neben zwei gut studierten Gruppen innerhalb der Beta- und Gamma-Proteobakterien auch manche Archaea zur Ammoniak-Oxidation befähigt sind hat unser Verständnis der funktioniellen Kapazitäten dieser Domäne fundamental verändert. Weiters hat sie grundlegende Fragen zu der Verteilung, Abundanz und Aktivität der unterschiedlichen Ammoniak-oxidierenden Gruppen in der Umwelt aufgeworfen. Innerhalb der letzten Jahre wurde gezeigt, dass Ammoniak-oxidierende Archaeen (AOA) die bis dahin bekannten Ammoniak-oxidierenden Bakterien (AOB) in vielen Systemen in Bezug auf ihre numerische Abundanz bei weitem übertreffen. Weiters konnten molekulare Studien die Präsenz von amo-Genen die für das Schlüsselenzym Ammoniak-Monooxygenase codieren in vielen unterschiedlichen Habitaten nachweisen. Es bleibt jedoch zu zeigen, ob Ammoniak tatsächlich das natürliche Substrat dieser Enzyme, die typischerweise eine Vielzahl von Substraten umsetzen können, darstellt. Ein limitierender Faktor ist sicherlich die sehr geringe Zahl der derzeit verfügbaren Anreichungs- bzw. Reinkulturen von AOA, die zur Aufklärung solcher Fragen dienen könnten. Kürzlich zeigten Genom-vergleichende Studien eines „marinen Gruppe I.1a“ Archaeons, „Candidatus Cenarchaeum symbiosum“, das dieser Organismus nicht ein Mitglied eines der etablierten Phyla der Archaea ist, sondern tatsächlich in ein neues, bisher unbekanntes Phylum, das nun als Thaumarchaeota bezeichnet wird, fällt. Als diese Studie durchgeführt wurde war allerdings nur ein einziges Genom eines Thaumarchaeoten verfügbar, was anfangs zu einer starken Zurückhaltung der Wissenschaftsgemeinde gegenüber der Idee eines neuen Phylums der Archaea führte. Meine Doktorarbeit umfasste fünf thematisch eng miteinander verknüpfte Projekte, die sich mit Ammoniak-oxidierenden Mikroorganismen beschaftigten: Das Kernprojekt widmete sich der Identifizierung eines moderat thermophilen Archaeons aus einer Anreicherungskultur einer heißen Quelle als Ammoniak-Oxidierer und dessen Charakterisierung. Dieser AOA, einstweilen als „Candidatus Nitrososphaera gargensis“ klassifiziert, stellt den ersten kultivierten Vertreter der global verteilten „I.1b Boden-Gruppe“ der Archaea dar. Es wurde gezeigt, dass die metabolische Aktivität dieses Archaeons durch die Konzentration des verfügbaren Ammoniaks beeinflusst wird. So ist diese Mikrobe bei niedrigen Ammoniak-Konzentrationen hochaktiv, während sie bereits durch leicht erhöhte Konzentrationen, die noch keinen hemmenden Effekt auf bekannte AOB haben, inhibiert wird. Diese Entdeckung, dass AOA an niedrige Ammoniakkonzentrationen angepasst sind, wurde seither durch andere Gruppen bestätigt und erweitert und erklärt zum Teil die Nischen-Differenzierung zwischen AOA und AOB in der Umwelt. In einer Folgestudie wurde basierend auf den Genomsequenzen von N. gargensis, C. symbiosum und „Candidatus Nitrosopumilus maritimus“ die Thaumarchaeota-Hypothese mittels vergleichender Genomik überprüft. Es konnte gezeigt werden, dass verschiedene genomische Charakteristika und phylogenetische Analysen die Besonderheit und ursprüngliche Position der Thaumarchaeota innerhalb der Domäne der Archaea unterstützen. Da dieses Phylum offenbar die zahlenmäßig bedeutendste Linie der Archaea auf unserem Planeten darstellt, fällt dem Studium des funktionellen und genetischen Potentials der Mitglieder dieser Gruppe eine besondere Bedeutung zu. Die Untersuchung einer Anreicherungskultur, die mit Material aus einer heißen Quelle auf der Halbinsel Kamtschatka beimpft wurde, führte zur Identifikation von „Candidatus Nitrosotenuis uzonensis“. Dieser Organismus stellt den ersten moderat thermophilen AOA innerhalb der Gruppe I.1a der Thaumarchaeota dar. Mit Hilfe von Metagenomik-Techniken wurde nahezu das komplette Genom des AOA sequenziert und erste Einblicke in die Biochemie der Ammoniak-Oxidation und Kohlenstoff-Fixierung des Organismus erhalten. Weiters wurde gezeigt, dass dieses Archaeon ein Repräsentant einer in terrestrischen, heißen Habitaten global verbreitenden Linie von Ammoniak-Oxidierern ist. Weiters war ich an einer Studie über die Rolle von unkultivierten Thaumarchaeoten in europäischen Kläranlagen beteiligt. Zum jetzigen Zeitpunkt ist Ammoniak-Oxidation die einzige bekannte Physiologie dieses Phylums und der Ausdruck AOA wird daher häufig als Synonym für diese Gruppe verwendet. Es wurde gezeigt, dass in einer dieser Kläranlagen Thaumarchaeoten, die amoA Gene im Genom tragen, um mehrere Größenordnungen abundanter sind als AOB. Entgegen der Lehrmeinung gewinnen diese Archaeen allerdings nicht den Großteil der für ihre Vermehrung benötigten Energie aus der Oxidation von Ammoniak, sondern aus der Oxidation von noch nicht näher identifizierten organischen Substraten. Dieser Befund zeigt, dass amoA-Gene ein schlechter Marker für den Nachweis und die Quantifizierung von AOA in der Umwelt sind und zeigt dass Thaumarchaeoten andere Substrate als Ammoniak als Elektronendonor verwenden können. In meinem letzten Projekt widmete ich mich der physiologischen Charakterisierung zweier neuer Spezies der Beta-Proteobakterien die in zwei Ammoniak-oxidierenden Anreicherungskulturen aus russischen thermalen Quellen stark angereichert sind. Für beide Kulturen konnte gezeigt werden, dass AOA für die Ammoniak-Oxidation verantwortlich zeichnen, aber manche Daten deuteten auf eine mögliche Beteiligung der Betaproteobakterien an diesem Prozess hin. Das nahezu komplette Genom eines dieser Mikroben wurde mittels Metagenomik sequenziert. Es zeigt, dass die beiden Enzyme, die in AOA und AOB für zentrale Schritte der Ammoniak-Oxidation verantwortlich zeichnen, in diesem Organismus nicht kodiert sind. Die möglicherweise Ammoniak-oxidierende Aktivität dieser Betaproteobakterien unter autotrophen Bedingungen wurde durch die Kombination von Isotopen-Markierungs- mit in situ Hybridisierungs-Techniken untersucht. Nach über fünf Jahren Forschung kann für dieses Projekt leider kein abschließendes Ergebnis präsentiert werden. Es konnten jedoch Theorien zur Energiegewinnung dieser Bakterien entwickelt werden, die eine wichtige Basis für zukünftige Experimente darstellen.Nitrification, the biological aerobic oxidation of ammonia via nitrite to nitrate, is an essential step of the global biogeochemical nitrogen cycle. The recent finding that some archaea, besides two long-known bacterial groups within the beta- and gamma-subclasses of Proteobacteria, are capable of ammonia oxidation radically changed our perception of the functional capabilities of members of this domain. Furthermore, it raised fundamental questions regarding the distribution patterns, abundances and activities of the different ammonia-oxidizing lineages in those ecosystem where nitrification occurs. Within the last few years, ammonia-oxidizing archaea (AOA) have been demonstrated to outnumber ammonia-oxidizing bacteria (AOB) in many systems and molecular analyses revealed the presence of a huge diversity of archaeal amo genes, encoding the key enzyme ammonia-monooxygenase, in many different environments. However, as monooxygenases of the Amo type are substrate promiscuous it remains to be shown whether the actual substrate of all these archaeal amoA-like proteins is ammonia. Furthermore, due to the difficulties of cultivating the slow-growing AOA only a very limited number of enrichment or pure cultures that could be used to elucidate such and other questions regarding the physiology of these organisms are currently available. Interestingly, recent comparative genomics-based inferences of a member of the amo-encoding marine group I.1a archaea, “Candidatus Cenarchaeum symbiosum”, demonstrated that this organism is profoundly different to the well-established archaeal phyla, and it has thus been proposed that it belongs to a novel phylum named Thaumarchaeota. However, as this study was based on only one thaumarchaeotal genome (no more genomes from AOA were available at that time), the scientific community was still reluctant to accept the idea of a novel archaeal phylum. My Ph.D. studies included five tightly linked projects dealing with ammonia-oxidizing microbes. The core project dealt with the identification of a moderately thermophilic archaeon from a hot spring enrichment culture as an ammonia oxidizer and on its initial genetic and physiological characterization. This AOA, provisionally classified as “Candidatus Nitrososphaera gargensis”, represents the first cultivated member of the globally distributed soil group I.1b of archaea. It was shown that the metabolic activity of this AOA is influenced by the available ammonia concentration. At low substrate concentrations it is highly active and autotrophically fixes bicarbonate, while slightly elevated ammonia concentrations, known to have no inhibitory effect on any known AOB, lead to inhibition of autotrophy in this archaeon. The finding that AOA are adapted to low ammonia concentrations was later confirmed and extended by other research groups and at least partially explains the niche differentiation between AOA and AOB. Based on comparative genomics of N. gargensis, C. symbiosum and “Candidatus Nitrosopumilus maritimus” I contributed to the reinvestigation of the Thaumarchaeota-hypothesis. Various genomic features and phylogenetic analyses strongly support both the distinctiveness and ancestral position of the Thaumarchaeota. As this phylum might represent the most abundant archaeal lineage on Earth it is of obvious importance to thoroughly study the functional and genetic potential of its members. The investigation of an enrichment culture seeded with material from a Kamchatkan thermal spring, led to the identification of “Candidatus Nitrosotenuis uzonensis”. It represents the first cultured moderately thermophilic AOA affiliated to group I.1a thaumarchaeotes. A near-complete genome of this AOA was obtained via metagenome sequencing and allowed initial insights into the organism’s ammonia oxidation biochemistry and carbon fixation pathway. In addition, it could be demonstrated that this archaeon is a representative of a lineage of AOA globally distributed in terrestrial geothermal environments. Furthermore, I was involved in a study aimed at elucidating the role of uncultivated thaumarchaeotes in a range of European waste water treatment plants. So far, ammonia oxidation is the only known physiology for this phylum and the term AOA is thus sometimes used as synonym for this lineage. In the presented study it is demonstrated that in one of the analyzed industrial plants in which amoA-carrying thaumarchaeotes outnumber AOB by several orders of magnitude, these putative AOA obtain their energy for growth not by ammonia oxidation, but rather by oxidizing yet unknown organic substances. This unexpected finding highlights that archaeal amoA genes are an insufficient proxy for detecting and quantifying AOA in the environment and that the Thaumarchaeotes harbour members that use other electron donors than ammonia for energy generation. In the last project I focussed on the physiological characterization of two novel species of betaproteobacteria that were highly enriched in two ammonia-oxidizing enrichment cultures recently obtained by a collaboration partner from two Russian geothermal springs. In both of these cultures AOA could be demonstrated to contribute to the oxidation of ammonia, but circumstantial data suggested that the betaproteobacteria might also be participating in this process. The near-complete genome of one of these microbes was obtained via metagenomic sequencing and demonstrated that the organism does not encode enzymes known to be involved in the oxidation of ammonia by recognized AOA or AOB. The potential ammonia-oxidizing activity of these bacteria was tested via the combination of isotope-labelling techniques with in situ hybridization techniques. After more than five years of research on this project still no conclusive result can be presented. However, theories on the potential energy conservation pathways of the organisms were developed, which provide a basis for future experiments

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought

Effect of nitrification inhibitors on the growth and activity of Nitrosotalea devanaterra in culture and soil

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Common principles and best practices for engineering microbiomes

Despite broad scientific interest in harnessing the power of Earth's microbiomes, knowledge gaps hinder their efficient use for addressing urgent societal and environmental challenges. We argue hat structuring research and technology developments around a design-build-test-learn (DBTL) cycle will advance microbiome engineering and spur new discoveries on the basic scientific principles governing microbiome function. In this Review, we present key elements of an iterative DBTL cycle for microbiome engineering, focusing on generalizable approaches, including top-down and bottom-up design processes, synthetic and self-assembled construction methods, and emerging tools to analyze microbiome function. These approaches can be used to harness microbiomes for broad applications related to medicine, agriculture, energy, and the environment. We also discuss key challenges and opportunities of each approach and synthesize them into best practice guidelines for engineering microbiomes. We anticipate that adoption of a DBTL framework will rapidly advance microbiome-based biotechnologies aimed at improving human and animal health, agriculture, and enabling the bioeconomy

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for L-methionine, followed by fluorescent labeling of AHA containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater, and anoxic sediment. We also developed combined assays that couple BONCAT with rRNA-targeted FISH, enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labeling by nanoSIMS (^(15)NH_4^+ assimilation) for individual cells within a sediment sourced enrichment culture showed concordance between AHA positive cells and ^(15)N enrichment. BONCAT-FISH offers a fast, inexpensive, and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single cell level

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

This paper describes a microfluidics-based workflow for genetically targeted isolation and cultivation of microorganisms from complex clinical samples. Data sets from high-throughput sequencing suggest the existence of previously unidentified bacterial taxa and functional genes with high biomedical importance. Obtaining isolates of these targets, preferably in pure cultures, is crucial for advancing understanding of microbial genetics and physiology and enabling physical access to microbes for further applications. However, the majority of microbes have not been cultured, due in part to the difficulties of both identifying proper growth conditions and characterizing and isolating each species. We describe a method that enables genetically targeted cultivation of microorganisms through a combination of microfluidics and on- and off-chip assays. This method involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a “chip wash” technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures. We validated this targeted approach by cultivating a bacterium, here referred to as isolate microfluidicus 1, from a human cecal biopsy. Isolate microfluidicus 1 is, to our knowledge, the first successful example of targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project’s “Most Wanted” list, and, to our knowledge, the first cultured representative of a previously unidentified genus of the Ruminococcaceae family

Formate-Dependent Microbial Conversion of CO2 and the Dominant Pathways of Methanogenesis in Production Water of High-temperature Oil Reservoirs Amended with Bicarbonate

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Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors

In this study, dideoxy sequencing and 454 high-throughput sequencing were used to analyze diversities of the ammonia monooxygenase (amoA) genes and the 16S rRNA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in six municipal wastewater treatment plants. The results showed that AOB amoA genes were quite diverse in different wastewater treatment plants while the 16S rRNA genes were relatively conserved. Based on the observed complexity of amoA and 16S rRNA genes, most of the AOB can be assigned to the Nitrosomonas genus, with Nitrosomonas ureae, Nitrosomonas oligotropha, Nitrosomonas marina, and Nitrosomonas aestuarii being the four most dominant species. From the sequences of the AOA amoA genes, most AOA observed in this study belong to the CGI.1b group, i.e., the soil lineage. The AOB amoA and 16S rRNA genes were quantified by quantitative PCR and 454 high-throughput pyrosequencing, respectively. Although the results from the two approaches show some disconcordance, they both indicated that the abundance of AOB in activated sludge was very low