223 Methanotrophic flexibility of “Ca. Methanoperedens” and SRB in meromictic Lake Cadagno Presence of Mobile Genetic Elements (MGEs) in Lake Cadagno’s “Ca. Methanoperedens” To assess the metabolic flexibility and putative horizontal gene transfer (HGT) in the Lake Cadagno “Ca. Methanoperedens” MAGs, we examined the presence of ECEs. In this regard, we addressed the presence of Borgs, which have been described as novel giant ECEs, that are not classifiable as virus or plasmid. They have been shown to be associated with “Ca. Methanoperedens” and have propensity to assimilate genes that harbor potential for key metabolic pathways such as anaerobic methane oxidation, extracellular electron transfer, or stress resistance (Al-Shayeb et al., 2022; Schoelmerich et al., 2024). More recently, 40 unique putative Borgs markers have been described (Schoelmerich et al., 2024), which we employed against our “Ca. Methanoperedens” MAGs. Our search resulted in 16 out of 30 family domain putative Borg marker proteins (Supplementary Table 13 and 14), suggesting the absence of previously described Borg ECEs in our samples. We also explored additional mobile genetic elements (MGEs) belonging to the “Ca. Methanoperedens” MAGs (Supplementary Tables 15-17). Our search identified 64 contigs as potential MGEs (Supplementary Table 18). To assess whether any of these were viral, we screened for specific structural proteins (e.g., capsids) but found no positive hits (Supplementary Tables 19-21). We then looked for alternative signature proteins, including integrases and transposases, detecting seven and six hits, respectively (Supplementary Tables 18-21). Finally, in those integrases and transposases harboring contigs, we checked for the presence of terminal repeats, an indicator of MGE completeness, and identified two instances in “Sed MAG Methanoperedens 1” (contig_3571) and “Sed MAG Methanoperedens 2” (contig_21328) (Supplementary Table 18). We also evaluated the possible role of MGE in HGT of key genes associated with the adaptation of SRB into a syntrophic partnership with ANME, as proposed by Murali et al. (2023). However, proteins encoding structures responsible for aggregate formation between ANME and SRB (Supplementary Table 9) were not identified in any of the 64 potential MGEs investigated (Supplementary Table 18). This suggests that MGEs do not influence the establishment of this partnership. 7
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