Skip to main content

ORIGINAL RESEARCH article

Front. Microbiol.
Sec. Microbial Physiology and Metabolism
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1483703

Fractionating proteins with nitrite-reducing activity in "Candidatus Kuenenia stuttgartiensis" strain CSTR1

Provisionally accepted
Emea Okorafor Ude Emea Okorafor Ude 1Pranathi Sure Pranathi Sure 1Rimjhim Rimjhim Rimjhim Rimjhim 1Lorenz Adrian Lorenz Adrian 1,2Chang Ding Chang Ding 1*
  • 1 Helmholtz Centre for Environmental Research, Helmholtz Association of German Research Centres (HZ), Leipzig, Germany
  • 2 Technical University of Berlin, Berlin, Brandenburg, Germany

The final, formatted version of the article will be published soon.

    The anammox bacteria "Candidatus Kuenenia stuttgartiensis" are able to gain energy by combining ammonium and nitrite to form nitrogen gas, an ecologically and technically important activity. In this reaction, nitric oxide is a confirmed nitrite reduction intermediate that is subsequently combined with ammonium to produce hydrazine. However, the enzyme that reduces nitrite to nitric oxide remains elusive. Here we investigated the nitrite-reducing activity in "Ca. Kuenenia stuttgartiensis" strain CSTR1 to identify candidates for such an enzyme. An in vitro assay for nitrite-reducing activities was established and optimized, with which we followed the activity in protein fractions yielded by various fractionation methods. Separation of the cell extract of strain CSTR1 with size exclusion chromatography yielded active fractions corresponding to a molecular size range of 150-200 kDa. Several proteins coeluted with the nitrite-reducing activity, including the hydroxylamine dehydrogenase HOX, an NADP-dependent isopropanol dehydrogenase (Adh), an electron-transfer 4Fe-4S subunit protein (Fcp), and a nitric oxide detoxifying flavorubredoxin (NorVW). Further separation of the cell extract with anion exchange chromatography, however, resulted in much lower activity yields, and activities were distributed among several fractions. In addition, fractionation of cell extracts using ultracentrifugation and ultrafiltration linked the activity to HOX, but could not exclude involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism behind nitrite reduction in "Ca. Kuenenia" strains is more complex than presently described in the literature. Nitrite reduction appears to be strongly linked to HOX but may additionally require the involvement of other proteins.

    Keywords: Shotgun Proteomics, Fractionation, Anammoxosome, planktonic cultivation, Nitrogen Cycle

    Received: 20 Aug 2024; Accepted: 28 Jan 2025.

    Copyright: © 2025 Ude, Sure, Rimjhim, Adrian and Ding. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Chang Ding, Helmholtz Centre for Environmental Research, Helmholtz Association of German Research Centres (HZ), Leipzig, Germany

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.