199 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress our enrichment ran as a sequencing batch reactor (SBR), planktonic cells are likely to be washed out of the system with regular exchange of the supernatant media (Supplementary Fig. 1B). The bioreactor system employed might explain why we observed such low signals of planktonic cells in our micrographs (Supplementary Fig. 12). The “Ca. Methanoperedens” investigated in this study was linked to the diminishment of PHA upon salt exposure (Fig. 5C). These observations align with recent observations on PHA decrease in a N-DAMO brackish enrichment exposed to marine salinities (Frank et al., 2023). Furthermore, we described the presence of three different ecologically underexplored sialic acids in our enrichment as well as their putative role upon salt-stress. Our “Ca. Methanoperedens” MAG encoded the biosynthesis pathway for the full production of one of them, legionaminic acid, which has previously been described in the halophilic archaeon Halorubrum sp. PV6 (Zaretsky et al., 2018) (Fig. 6B). To conclude, we report the metabolic and physiological response of freshwater anaerobic methanotroph “Ca. Methanoperedens Vercelli Strain 1” to salinity increase. Although not primarily found in marine environments (Supplementary Fig. 2 and Supplementary Table 2), we show that “Ca. Methanoperedens” could tolerate a wide range of salinities, from freshwater to marine conditions. We identified the production of the key osmolyte N(ε)-acetyl-β-L-lysine and linked it to “Ca. Methanoperedens”. Furthermore, we expanded the known phylogenomic distribution of the key biosynthetic kamA and ablB genes-encoding proteins across an archaeal-focused universal evolutionary tree. We propose that these genes may have been horizontally transferred from the bacterial phylum Firmicutes to the ANME archaea and halophilic methanogen Methanosalsum. We further reveal the putative roles of carbon storage polymers (PHAs) and extracellular polysaccharide structures as coping mechanism to salt stress in “Ca. Methanoperedens”. Together, this information may help to understand how the anaerobic methane filter in coastal ecosystems will respond to predicted environmental pressures in the future. To further enhance the understanding of natural saline intrusions, the introduction of marine taxa to freshwater 6
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