210 Chapter 7 v1.5.0 (Shaffer et al., 2020) with default options, except min_contig_size at 1000 bp, and METABOLIC v4 (Zhou et al., 2022). Additionally, we searched for genes of interest in the annotation files via BLASTp, applying an e-value cut-off of 10-5. For a more accurate functional prediction of hydrogenases, we used the curated HydDB classifier http://services.birc.au.dk/hyddb/ (Søndergaard et al., 2016). To corroborate poorly annotated genes/proteins, manual curations were validated with the NCBI Batch Entrez Conserved Domains and InterPro (Blum et al., 2021). In this study, we consider microorganisms as “canonical SRBs” if they encode the DsrD subunit of the dissimilatory sulfite reductase. This subunit has been identified as a marker for the enzyme’s reductive directionality, specifically for the reduction of sulfite to hydrogen sulfide (Diao et al., 2023). The search for proteins involved in SRB and ANME aggregate formation was done using DIAMOND with a list of protein InterPro IDs recovered from Murali et al., 2023 (IPR039662, IPR053783, IPR025295, IPR035903, PF01833) with e-value and identity cut-offs of 10-5 and 30%, respectively. Average amino acid or nucleotide identity (AAI or ANI) from MAGs was obtained using the FastAAI or FastANI-matrix tool option (Rodriguez-R & Konstantinidis, 2016), using 90% AAI or 95% ANI for species cutoff (Jain et al., 2018; Konstantinidis et al., 2022). Multi-heme c-type cytochrome (MHC) search and domain tree We used the assembled metagenomes to investigate the presence and type of MHC on the five “Ca. Methanoperedens” MAGs recovered together with the suspected syntrophic SRB “Desulfobacterota class QYQD01”. We first screened the selected MAGs for putative MHCs, identified by ORFs with ≥3 CXXCH motifs and used FeGenie v1.2 (Garber et al., 2020) annotations to differentiate Omcz nanowires, and BLASTp to identify Extracellular Cytochrome Nanowires (ECN). Subsequently, we used InterProScan v5.44-79.0 (Jones et al., 2014) to identify domains classified as “multiheme cytochromes” from the identified “Ca. Methanoperedens” MHC-harboring proteins. For this analysis, we also included reference bioreactor enrichment MAGs of “Ca. Methanoperedens” with confirmed metatranscriptomic evidence for MHC expression under various conditions, including: “Ca. Methanoperedens ferrireducens” (Fe-AOM) (Cai et al., 2018), “Ca. Methanoperedens manganicus (Mn1)” and “Ca. Methanoperedens manganireducens (Mn-2)” (Mn-AOM) (Leu et al., 2020a), “Ca. Methanoperedens nitroreducens” Type Strain (electrode/nitrate/iron-
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