Maider Junkal Echeveste Medrano

227 Methanotrophic flexibility of “Ca. Methanoperedens” and SRB in meromictic Lake Cadagno and “Ca. Methanoperedens” spp. N1.45 and S2.57 (Figure 3). For the here described putative syntrophic SRBs, we recovered cytosolic NiFe group 3c hydrogenases. Murali et al. (2023) assembled widespread syntrophic marine SRB clades that partner with ANME to perform S-AOM and identified traits that might be suggestive of an adaptation to a syntrophic lifestyle. Here, additional features indicative of syntrophy were highlighted, such as the ability for biofilm formation, intercellular communication, or for some Seep-SRB1a, a nutritional dependency on ANME based on the lack of a cobalamin synthesis pathway. Our study’s Desulfobacterota class QYQD01 clustered closely to marine syntrophic group Seep-SRB1g in the presented Desulfobacterota genome tree (Murali et al., 2023b; Skennerton et al., 2017). For this comparative genomics study, Desulfobacterales family ETH-SRB1 (refer to as SeepSRB1a sp.1) was also considered as a syntrophic SRB, aligning with the presented sulfate-AOM in co-abundance with “Ca. Methanoperedens’s spp. N1.45 and S2.57 in Olkiluoto Island deep subsurface in Finland (Bell et al., 2022; Murali et al., 2023b). Our investigation into ECEs in “Ca. Methanoperedens” produced inconclusive results regarding the presence of Borgs. Specifically, the markers used to identify Borgs (Schoelmerich et al., 2024) did not provide clear evidence of their presence in the recovered genomes. Only 17 out of the 30 Borg family protein markers were detected (Supplementary Tables 13 and 14) so the presence of previously identified Borgs seems unlikely. Whether those 17 Borg family marker proteins belong to as of yet unidentified Borgs needs to be further investigated. Additionally, only two non-viral MGE belonging to “Sed MAG Methanoperedens 1” and “Sed MAG Methanoperedens 2” were conclusively identified (Supplementary Table 18). To conclude, we describe an ubiquitous co-abundance of “Ca. Methanoperedens” spp. and Desulfobacterota class QYQD01 to sustain sulfate-AOM in sulfate-rich freshwater systems, mainly present in groundwater and marine systems. We suggest putative MHC-proteins of “Ca. Methanoperedens” that could engage via EET in a syntrophic interaction and present metabolic adaptations and phylogenomic placement of Desulfobacterota indicative of a syntrophic lifestyle with ANME archaea. Future efforts should focus on S-AOM-targeted enrichments of “Ca. Methanoperedens” and syntrophic SRB in bioreactors inoculated with sediment 7

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