Membrane complexes involved in anaerobic ammonium oxidizers

• Applicants: Dr. Thomas Barends; Dr. Kristian Parey
• Subject Area: Microbial Ecology and Applied Microbiology ; Metabolism, Biochemistry and Genetics of Microorganisms ; Structural Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 514060533

Anaerobic ammonium-oxidizing (“anammox”) bacteria are one of the latest additions to the biogeochemical nitrogen cycle and can be found worldwide. These extraordinary microorganisms with an unusual morphology derive their energy from the oxidation of ammonium coupled with nitrite reduction that relies on highly toxic intermediates such as hydrazine and nitric oxide. About 50% of the released dinitrogen gas is produced by anammox bacteria. In biotechnology, the anammox process is used as a sustainable alternative to current wastewater treatment systems to remove nitrogen compounds. We have made significant contributions to elucidate the nature of the catabolic pathway and to characterize the key soluble enzymes. However, very little is known about the membrane complexes at the heart of this bioenergetic process and how the proton gradient is established across the anammoxosomal membrane that drives ATP synthesis. With this proposal, we will address the nature of membrane complexes by leveraging the synergy between electron cryo-microscopy (cryo-EM), crystallography, biochemistry, and electrochemistry to open a new avenue to understand anammox bioenergetics. Our work program is (i) to purify bc1 complexes and demonstrate their function by turn-over experiments and proton-pumping activity assays. Structural studies by single-particle cryo-EM will shed light on the mechanism of how the ubiquinone pool feeds the bc1 complexes and the proton gradient is built up; (ii) to address how electrons are transferred within the NXR system. For this purpose, the NXRmem complex will be purified by chromatography for cryo-EM studies. In parallel, the various components of this complex will be heterologously expressed for biochemical and electrochemical studies; and (iii) to use cryo-electron tomography, sub-tomogram averaging, and segmentation to study the molecular topology of the membranes of anammox bacteria. This technique will provide insights into the native landscape at molecular resolution and bioenergetics of this largely unexplored organism. In particular, protein complexes in the respiratory chain can be differentiated and their organization examined in situ.

• DFG Programme: Research Grants
• International Connection: Netherlands
• Cooperation Partner: Professorin Dr. Laura van Niftrik