Metabolic an Genomic Studies of Ammonia-Oxidizing Archaea

Nitrification is a process solely performed by microorganisms, that plays a central role in the global cycling of nitrogen and is of economic importance in agriculture and wastewater treatment. The first step in nitrification is performed by ammonia oxidising microorganisms that convert ammonia into nitrite ions. Thanks to their discovery by S. Winogradsky, nitrifying bacteria have been known for more than hundred years. Only recently, it has been recognized that microorganisms of the domain archaea are also able to perform this process. This group of archaea outnumber the known bacterial nitrifiers by orders of magnitudes in the marine plankton, as well as in many soils, sediments and estuarines. Although present in very large numbers, very little is known about the physiology of ammonia oxidizing archaea. Their chemolithoautotrophic growth mode has so far been shown only for a single cultivated isolate from a marine aquarium and for two enrichments from hot environments. Therefore, the physiology of ammonia oxidizing archaea in particular of those from soil has remained elusive and their contribution to nitrification has been debated. We have recently isolated a chemolithoautotrophic ammonia oxidizing archaeon from a garden soil in Vienna that is stably growing in enrichment cultures for two years and is enriched up to >95%. The overall goal of this proposal is to get a deeper insight into the physiology, general activities, evolution and genomic potential of Candidatus Nitrososphaera viennensis and thus to develop it into a model organism for ammonia oxidizing archaea from soil. For this purpose we will perform detailed physiological characterisations, as well as genomic and functional genomic studies. We will attempt to get a pure culture from one of the two strains that we have enriched and will explore its metabolic diversity, exploring the use of different electron donors and acceptors, its sensitivity to inhibitors, and its emission of nitrous oxide. Starting from a draft genome that has already been obtained, we will close the genome sequence and complement our annotation efforts, also employing transcriptomics and some (limited) proteomic studies, that will give us insights into the most abundantly expressed proteins. The complete genome sequence as well as metabolomic studies will also assist in reconstructing major metabolic pathways of the organism. In co-cultures of ammonia oxidizing archaea and bacteria, we will explore and compare the activity and transcriptional patterns of the different ammonia oxidizers in order to get insights into the differences of their ecophysiology. In total we expect to get a deeper insight into this widely distributed and potentially ecologically very significant group of archaea.

Thaumarchaeota occur in huge numbers in almost all aquatic and terrestrial environments on Earth. Members of this phylum of the Archaea gain energy by oxidizing ammonia, i.e. performing the first step of nitrification, which is a fundamental process of the nitrogen cycle on Earth. Due to the rising human population with an increasing food demand, extensive applications of nitrogen fertilizer in agriculture is common practice. As this severely effects soil microbial communities and increases greenhouse gas emissions, field studies on the activity and abundance of ammonia-oxidizing archaea (AOA) compared to their bacterial counterparts (AOB) under various environmental parameters have been performed. However, the isolation and cultivation of AOA has proven to be difficult.The aim of this project was to obtain the first pure culture of an ammonia oxidizing Thaumarchaeote from soil, to study its metabolism and morphology in detail and to analyze its genome and compare it to other related organisms. The overall goal of this attempt was to improve our knowledge on the physiology of this group of nitrifiers that are very little known, yet are abundant in all terrestrial ecosystems and have a potentially important role in pristine and anthropogenic environments. Nitrososphaera viennensis was isolated and purified from a Viennese garden soil. It is an aerobic, neutrophilic organism that produces energy by oxidizing ammonia to nitrite. It grows predominantly autotrophically by fixing CO2 to build up biomass. However, the organism is strongly dependent on the addition of carboxylic acids in order to sustain a generation time of approximately 29 hours. N. viennensis can use urea instead of ammonia as an alternative energy source. During ammonia oxidation it produces traces of the potent greenhouse gas nitrous oxide in a hybrid formation mechanism, but not under reduced oxygen concentrations like its bacterial counterparts. N. viennensis is a small, irregular coccus that is flagellated and has a proteinaceous surface layer with p3-symmetry, as known for hyperthermophilic archaea of the order Sulfolobales. The genomes of N. viennensis and it close relative N. gargensis reveal typical cellular features of other archaea and exhibit fundamental differences between the central metabolisms of ammonia oxidizing archaea and bacteria. They also reveal potential alternative carbon and energy sources used by AOA.This study provides a basis for future detailed physiological and functional genomic analyses aimed to understand the so far unresolved ammonia oxidation pathway of these organisms, to obtain a deeper understanding of their niche adaptations, their importance in the biogeochemical nitrogen cycle and to develop strategies to mitigate greenhouse gas emissions.