Investigating the influence of nitrogen cycling on climate change, one enzyme at a time

Secondary Supervisor(s): Professor David Roper

University of Registration: University of Warwick

BBSRC Strategic Research Priority: Understanding the Rules of Life (Microbiology)

Project Outline

Ammonia oxidising microorganisms are crucial to the global nitrogen cycle and they play a significant role in food security and climate change. Ammonia oxidation is the first and rate-limiting step in nitrification and carried out by bacterial and archaeal ammonia oxidisers. Nitrification results in the emission of the potent greenhouse gas nitrous oxide and in the production of nitrate, which upon entering rivers and seas, causes eutrophication. Despite the functional importance, wide distribution, and vast global abundance of ammonia oxidising microorganisms, little is known about key enzymes responsible for ammonia oxidation. This is because the enzymes have not yet been purified in their active forms, and some of the novel enzymes have not yet even been identified. This project will use innovative click chemistry to study the enzymes central to global biogeochemical cycling, with focus on the ammonia monooxygenase (Fig. 1). Over the course of this project, you will generate new knowledge on the physiology, biochemistry, and ecology of microorganisms, which are crucially important for global biogeochemical cycling of nitrogen.

Aims

Figure 1. Click chemistry will be used to fluorescently label enzymes of interest as shown in the model ammonia oxidising archaeon 'Ca. Nitrosocosmicus franklandus C13'.

We have recently developed a new click chemistry method in collaboration with colleagues at Radboud University in the Netherlands, to label active monooxygenases, including those from ammonia oxidising archaea. This enables us to gain novel insights into subcellular localisation of these enzymes and study microbial populations in situ in the environment in greater detail that has been possible before. The project will make use of this technique to label monooxygenase enzymes. The aim of the project is to characterise the monooxygenases, to visualise enzymes within microbial cells and to retrieve active microbial populations from the environment to gain insights into how these key microorganisms perform their functions. Key questions you will address are: Where in the microbial cells are the enzymes located and how does this affect their function? Which microorganisms are actively cycling nutrients in the environment?

Methodology

You will start the project by using click chemistry to characterise enzymes in pure cultures of ammonia oxidising bacteria and archaea. These results will provide the basis for further experiments with complex, mixed microbial communities. The project will use an exciting combination of molecular and cultivation-based techniques in addition to click chemistry (Fig. 1), including fluorescence in situ hybridisation (FISH), flow cytometry and fluorescence-activated cell sorting (FACS), DNA-based methods such as PCR and sequencing, Western blotting, microscopy and whole cell enzyme kinetics assays.

Training

You will receive advice, support and subject-specific training from the supervisory team and the members of the Lehtovirta-Morley and Roper labs at the University of Warwick. This project will be carried out in collaboration with the Lücker lab at Radboud University and there will be an option to train and conduct research in the collaborator’s lab in the Netherlands You will present your work in regular lab meetings, seminars and (inter)national conferences. You will receive training in scientific and transferable skills as part of your cohort training and through courses offered at the University of Warwick. Students are also encouraged to attend seminars and engage in outreach.

The project will suit a student interested in microbiology, physiology, biochemistry and biogeochemical cycling. Applicants are strongly encouraged to contact Laura Lehtovirta-Morley (l.lehtovirta-morley@uea.ac.ukLink opens in a new window) to discuss the project and ask any questions they may have.

Further reading

Sakoula D, et al. (2022) Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria. ISME J 16, 958–971. Available at: https://doi.org/10.1038/s41396-021-01144-0Link opens in a new window 

Sakoula D, et al. (2024) Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea. ISME Commun 4(1), ycae092. Available at: https://doi.org/10.1093/ismeco/ycae092Link opens in a new window 

Wright CL, et al. (2020) Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes. Appl Environ Microbiol 86(9), e02388-19. Available at: https://doi.org/10.1128/AEM.02388-19Link opens in a new window 

Lehtovirta-Morley LE, et al. (2016) Isolation of ‘Candidatus Nitrosocosmicus franklandus’, a novel ureolytic soil archaeal ammonia oxidiser with tolerance to high ammonia concentration. FEMS Microbiol Ecol 92(5): fiw057. Available at: https://doi.org/10.1093/femsec/fiw057Link opens in a new window