211 Methanotrophic flexibility of “Ca. Methanoperedens” and SRB in meromictic Lake Cadagno AOM) (Zhang et al., 2023) and “Ca. Methanoperedens Vercelli” (electrode-AOM) (Ouboter et al., 2024). The identified MHCs domains were subtracted from the protein sequence and aligned using MAFFT v7.525 (Katoh & Standley, 2013) via https://www.ebi.ac.uk/ with parameters --bl 62 --op 1.53 --ep 0.123 --reorder --retree 2 --treeout --maxiterate 2 –amino. Finally, we constructed a phylogenetic tree using IQ-TREE v2.1.4 with function -s -st AA -m MFP -bb 1000 -nt AUTO, with a VT+R6 as best-fit model based on Bayesian Information Criterion (BIC). All presented trees were annotated using iTOL v5 (Letunic & Bork, 2021). Extrachromosomal elements (ECEs) search We additionally screened for putative ECEs in our five “Ca. Methanoperedens” MAGs. Among the ECEs investigated we included Borgs. For this, we first obtained a PFAM/InterPro database with dereplicated sequence representatives built from 40 different unique markers obtained from 17 different Borgs that appear to be associated with “Ca. Methanoperedens” (Schoelmerich et al., 2024). Then we used DIAMOND v2.1.9.163 (Buchfink et al., 2015) to perform a BLASTp query search against our “Ca. Methanoperedens” MAGs. For these annotations we applied an e-value cut-off of 10-5 and a minimum amino acid identity of 30%. We also searched for plasmids and viruses integrated into the “Ca. Methanoperedens” MAGs described in this study by using VIBRANT (Kieft et al., 2020) and geNomad (Camargo et al., 2024) with default parameters. Initially, we identified 68 contigs as potential ECEs. To further investigate the nature of contigs, we classified them using specialized viral and phage annotation databases, including PHROGs (Terzian et al., 2021) pVOGs (Grazziotin et al., 2017), and VOGs (Bao et al., 2004). The e-value and identity cut-offs used for this annotation were 10-5 and 30%, respectively. 7
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