158 Chapter 5 We also checked for additional intrinsic mechanisms related to sulfur cycling present in the N-DAMO partners (Figure 3) “Ca. Methanoperedens BLZ2” and “Ca. Methylomirabilis oxyfera”. This targeted annotation search assigned sulfate adenylyltransferase (sat) (KEGG: K00958) to both MAGs and L-cysteine S-thiosulfotransferase (soxX) (KEGG: K17223) only to MAG “Ca. Methylomirabilis oxyfera”. Regarding significant transcriptional shifts in S cycling genes, “Ca. Methylomirabilis oxyfera” showed the clearest significant increase on sat and soxX (Supplementary Table 5). Our study highlights the relevance of sulfite reductases upon low and long-term exposure to sulfide (Table 4). These enzymes are known to protect methanogens from sulfite inhibition (Balderston & Payne, 1976; Morrison & Mojzsis, 2021; Susanti & Mukhopadhyay, 2012). While sulfide gets incorporated into the biomass, sulfite builds up inside the cell reacting with nucleic acids, proteins, and enzyme co-factors (Schimz, 1980). We identified two significantly upregulated sulfite reductases belonging to the Group III Dsr-LP (IPR045169) exclusive to “Ca. Methanoperedens” and not present in marine ANME but in methanogens (Dalcin Martins et al., 2024, Chapter 2; Yu et al., 2018). Recently, combined transposon library construction with high-throughput growth studies on model methanogen Methanococcus maripaludis showed that the uncharacterized Group III-d Dsr-LP allowed for sulfite resistance in the presence of sulfite as a unique sulfur source (Day Leslie et al., 2024). On the contrary, marine ANME were hypothesized to employ sulfite reductase Group II coenzyme F420-dependent sulfite reductase (Fsr), a protein found to be more abundantly present in the metaproteome of marine ANME (Yu et al., 2018) or, hypothesized to confer sulfite detoxification potential in brackish ANME-2 (Dalcin Martins et al., 2024, Chapter 2). Still, Group II Fsr has not been found in “Ca. Methanoperedens” MAGs (Yu et al., 2018). Similar to Group III-d Dsr-LP, Group I Fsr was first described to be highly expressed under sulfite as sole sulfur source and provided detoxification potential in a Methanocaldococcus jannaschii culture (Johnson & Mukhopadhyay, 2005). The distinct presence of different groups of sulfite reductases in freshwater and marine ANME could explain differential acclimation to sulfide exposure.
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