191 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress We subdivided all differentially expressed transcripts into three different categories: central metabolism, cell morphology/locomotion, and overall stressrelated response. The methyl-coenzyme M reductase complex (MCR) genes had a lower relative expression at increased salinity (Fig. 4A). Besides, genes linked to nitrosative stress response (nitric oxide reductases norB and norC) increased in expression (Fig. 4A), in accordance with the accumulated nitrite and reduced ammonium production observed prior to sampling (Supplementary Fig. 1A). Transcripts encoding proteins involved in small amino acid biosynthesis with simpler molecular structure and smaller molecular weight, and other potential compatible solutes, in addition to N(ε)-acetyl-β-L-lysine, were also upregulated (argG_1, argG_2 and glmS; Fig. 4A). Morphology-associated transcripts were more abundantly expressed: genes encoding for the cell division machinery (ftsZ) and flagellar biosynthesis were upregulated (Fig. 4B). However, microscopy imaging was of too low resolution to visualize “Ca. Methanoperedens” morphotype shift from granules to free-living or planktonic cells (Supplementary Fig. 12). We also observed a general increase in stress-related transcripts, with a particular increase in genes encoding ABCtransporters (categorized in substrate transport), putative transposases (categorized as DNA replication repair), and cation transporters (Fig. 4C). Metatranscriptomic data indicated a downregulation of a large mechanosensitive (MS) channel (MScL)- encoding mscL (Fig. 4C). Another stress-related transcript was a gene encoding a heme-containing ferritin-like protein (belonging to the cd00657 family), and a non-heme containing ferritin-like AB (archaea and bacteria) (classified as cd01045 sub-family of ferritins), which were up- and downregulated, respectively (Fig. 4C). We also investigated additional potentially relevant extrachromosomal gene expression shifts upon salt stress. Our same study culture was previously described to lack Borgs, although two plasmids were found (Schoelmerich et al., 2022). In this study, we managed to retrieve one of the previously well-curated and predominant (highest coverage) plasmids, HMp_v2, from our metagenome (contig id: contigs_ NODE_75__2494.086047). This plasmid, composed of 148 Open Reading Frames (ORF), had 9 and 1 up and downregulated transcripts, respectively (Supplementary Table 8 and 10). Among the significantly downregulated genes, we identified a gene encoding a protein involved in cell motility (FlaI_TIVSS_p) (Fig. 4B). A gene 6
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