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Anaerobic ammonium oxidation (anammox) has received significant attention during optimization of waste-water treatment and constitutes an important pathway for the removal of bioavailable nitrogen from natural environments. Studies of key catabolic enzymes indicate that the anammox reaction takes place inside the anammoxosome, an organelle-like membranous compartment of anammox bacteria. The anammoxosome has also been suggested as a site for ATP synthesis. A lipid-based protein immobilization technique, previously used to identify proteins essential for the anammox reaction, was in this study used to select linear epitopes for antibodies specifically targeted against an identified ATPase. The approach of using proteomics and bioinformatics as tools for selecting antibody targets for immunolocalization provides an important alternative to traditional methods for selection of specific antibodies. Immunogold electron microscopy and statistical evaluations indicated that the antibodies against the ATPase were exclusively found associated with the anammoxosome membrane. This provides strong evidence for ATP synthesis by an intracellular proton motive force in anammox bacteria. Within prokaryotes, an ATP synthase associated with an intracellular compartment is a feature unique for anammox bacteria., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N(2). Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N(2) production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N(2) and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.