Development of tools for the study of enzymes in ammonia oxidising archaea

Schatteman, Arne (2022) Development of tools for the study of enzymes in ammonia oxidising archaea. Doctoral thesis, University of East Anglia.

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Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. In addition, AOA are hard to grow, and laboratory techniques are poorly developed. The main aim of this thesis was to identify the unknown proteins in the archaeal ammonia oxidation pathway and an additional goal was to improve the methods for growing and working with the model organism ‘Ca. N. franklandus’.

A bioreactor system was explored to grow ‘Ca N. franklandus’. A continuous cultivation system with biomass retention was shown to be a promising way to culture this organism. If successfully deployed, it would provide permanent access to high quality biomass in sufficient quantities for physiological experiments. In addition, a cell breakage protocol was optimised, and a proteome of cells grown on urea and ammonia was determined.

To investigate the ammonia oxidation pathway, substrates and inhibitors of the hydroxylamine oxidation mechanism were identified and characterised. Hydrazine and phenylhydrazine were shown to interfere with ammonia and hydroxylamine oxidation in AOA. Furthermore, ‘Ca. N. franklandus’ oxidized hydrazine into dinitrogen, coupling this reaction to ATP production and O2 uptake.

Furthermore, activity-based protein profiling (ABPP) probes were evaluated for the labelling of the ammonia monooxygenase and the hydroxylamine oxidation enzyme. To this end, 1,5-hexadiyne and an aryl-hydrazine probe, respectively, were evaluated. The diyne probe has been successfully used to label the AMO of AOA and the aryl probe shows promising results which may lead to the identification of the hydroxylamine oxidation enzyme.

Finally, several observations in this thesis led to the identification of a catalase isozyme. First, a DNA protection during starvation protein was very abundant in the proteome. Second, this protein was identified when a catalase staining method was used and catalase activity was observed while no catalase is encoded in the genome of ‘Ca. N. franklandus’. Sequence analysis supported the hypothesis that it was a DPS-like protein with catalase activity.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 26 Oct 2023 07:18
Last Modified: 26 Oct 2023 07:18

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