Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1
The anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in th...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-02-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1483703/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850078116069769216 |
|---|---|
| author | Emea Okorafor Ude Pranathi Sure Rimjhim Rimjhim Lorenz Adrian Lorenz Adrian Chang Ding |
| author_facet | Emea Okorafor Ude Pranathi Sure Rimjhim Rimjhim Lorenz Adrian Lorenz Adrian Chang Ding |
| author_sort | Emea Okorafor Ude |
| collection | DOAJ |
| description | The anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in the reduction of nitrite, which is subsequently combined with ammonium to produce hydrazine. However, the enzyme that converts nitrite to nitric oxide remains elusive. In this study, we investigated the nitrite-reducing activity in “Ca. Kuenenia stuttgartiensis” strain CSTR1 to identify candidates for such an enzyme. An optimized in vitro assay was established to measure nitrite-reducing activities, with which we followed the activity in protein fractions obtained from 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). However, further separation of the cell extract with anion exchange chromatography, 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 the involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism for nitrite reduction in “Ca. Kuenenia” strains is more complex than that currently described in the literature. Nitrite reduction appears to be strongly associated with HOX but may additionally require the participation of other proteins. |
| format | Article |
| id | doaj-art-5e57c59c80cc476b8f2e148f52f78a5c |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-5e57c59c80cc476b8f2e148f52f78a5c2025-08-20T02:45:38ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-02-011610.3389/fmicb.2025.14837031483703Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1Emea Okorafor Ude0Pranathi Sure1Rimjhim Rimjhim2Lorenz Adrian3Lorenz Adrian4Chang Ding5Department of Molecular Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, GermanyDepartment of Molecular Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, GermanyDepartment of Molecular Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, GermanyDepartment of Molecular Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, GermanyChair of Geobiotechnology, Technische Universität Berlin, Berlin, GermanyDepartment of Molecular Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, GermanyThe anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in the reduction of nitrite, which is subsequently combined with ammonium to produce hydrazine. However, the enzyme that converts nitrite to nitric oxide remains elusive. In this study, we investigated the nitrite-reducing activity in “Ca. Kuenenia stuttgartiensis” strain CSTR1 to identify candidates for such an enzyme. An optimized in vitro assay was established to measure nitrite-reducing activities, with which we followed the activity in protein fractions obtained from 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). However, further separation of the cell extract with anion exchange chromatography, 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 the involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism for nitrite reduction in “Ca. Kuenenia” strains is more complex than that currently described in the literature. Nitrite reduction appears to be strongly associated with HOX but may additionally require the participation of other proteins.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1483703/fullshotgun proteomicsfractionationanammoxosomeplanktonic cultivationnitrogen cycle |
| spellingShingle | Emea Okorafor Ude Pranathi Sure Rimjhim Rimjhim Lorenz Adrian Lorenz Adrian Chang Ding Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 Frontiers in Microbiology shotgun proteomics fractionation anammoxosome planktonic cultivation nitrogen cycle |
| title | Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 |
| title_full | Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 |
| title_fullStr | Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 |
| title_full_unstemmed | Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 |
| title_short | Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1 |
| title_sort | fractionating proteins with nitrite reducing activity in candidatus kuenenia stuttgartiensis strain cstr1 |
| topic | shotgun proteomics fractionation anammoxosome planktonic cultivation nitrogen cycle |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1483703/full |
| work_keys_str_mv | AT emeaokoraforude fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 AT pranathisure fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 AT rimjhimrimjhim fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 AT lorenzadrian fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 AT lorenzadrian fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 AT changding fractionatingproteinswithnitritereducingactivityincandidatuskueneniastuttgartiensisstraincstr1 |