159 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea Another area for future studies is the possible dual role of “Ca. Methanoperedens” Group III Dsr-LP not only as a sulfite reductase but also as nitrite detoxification mechanism. During our long-term exposure to sulfide, nitrite accumulated in the bioreactor (Supplementary Figure 1A).The simplest sulfite reductase structure, belonging to the Group I Fsr sulfite reductase, was extracted and crystalized in Methanothermococcus thermolithotrophicus together with follow up enzymatic assays indicating a higher nitrite (Km= ~ 2.5 µM) over sulfite (Km= ~ 15.5 µM) preference (Jespersen et al., 2023). Furthermore, Jespersen et al. (2023), analyzed the binding pocket for sulfite and observed that Group II Fsr has a larger binding pocket than the analyzed Group I Fsr. This observation led to the hypothesis that Group II Fsr might harbor a different substrate specificity. Similarly, in an attempt to characterize the catalytic activity of Group II Fsr in ANME, a purified recombinant ANME-2c expressed in Methanosarcina acetivorans was employed, leading to the discovery that no sulfite or thiosulfate activity occurred but instead Group II Fsr gave physiologically relevant nitrite reductase activity (Heryakusuma et al., 2022). All together, this suggested that a potential role of Group II Fsr could be conferring nitrite detoxification potential to non-nitrate reducing ANME. Concomitantly, if we extrapolate that observation “Ca. Methanoperedens”, no Group II Fsr in “Ca. Methanoperedens” have been described, which can alternatively employ the nitrite reductase NrfAH to perform DNRA (Arshad et al., 2015; Dalcin Martins et al., 2022, Chapter 4) and are therefore not dependent on an additional nitrite detoxification system. The described upregulated Group III Dsr-LP sulfite reductase gene is phylogenetically distinct to Fsr groups and most closely related to AsrC, which is distinct both from dissimilatory DsrAB but also aSir (Yu et al., 2018). AsrC has been described to work as a dissimilatory sulfite reductase (Huang & Barrett, 1991; Simon & Kroneck, 2013). Still, AsrABC has also been reported to be upregulated under nitrate, and not sulfate, amendment in the acidophilic sulfate reducer Acididesulfobacillus acetoxydans (Egas et al., 2024). In Egas et al. (2024), a novel putative dissimilatory nitrate-reducing enzyme - DEACI_1836 - classified as nitrite reductase [NAD(P)H]-like (IPR052034) and conserved hypothetical protein CHP03980, redox-disulfide (IPR023883) was described to act alone or together with AsrABC to perform DNRA. Still, no ANME or methanogens have been reported to 5
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