187 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress N(ε)-acetyl-β-L-lysine production pathway and biosynthetic operon organization N(ε)-acetyl-β-L-lysine is synthesized from L-Lysine (Fig. 2D). N(ε)-acetyl-β-L-lysine requires two enzymes for its synthesis: lysine-2,3-aminomutase (LAM, encoded by kamA) and β-lysine-N6-acetyl transferase (AT: encoded by ablB) (Fig. 2D). In our enrichment culture, the only ablB gene in the metagenome data was linked to the dominant “Ca. Methanoperedens Vercelli Strain 1” MAG, suggesting that this strain is responsible for its production (Supplementary Table 10). The kamA and ablB genes were present in the same operon together with a MscS-like ynaI gene, potentially encoding a small conductance mechanosensitive channel-like membrane protein (Fig. 2E). To accurately quantify kamA and ablB mRNA expression levels, we developed a targeted approach via reverse transcriptase (RT)-qPCR. A low baseline expression of both these genes was measured up to and including 1.5% salinity (Fig. 2F-G). Thereafter, the expression increased stepwise and was highest in the 3% salt conditions (Fig. 2F-G and Supplementary Fig. 9). These observations aligned with the microcosm experiments where “Ca. Methanoperedens” was unable to cope with a sudden salinity increase but was able to tolerate the increase when longer incubated allowing for the upregulation and production of the osmolyte N(ε)-acetylβ-L-lysine (Supplementary Fig. 3). Phylogenetic distribution suggests horizontal transfer (HGT) of genes encoding enzymes for N(ε)-acetyl-β-L-lysine synthesis To learn more about the phylogenetic distribution of N(ε)-acetyl-β-L-lysine production in ANME archaea, we retrieved KEGG-annotated kamA and ablB genes, which respectively encode the LAM and AT enzymes, from all genomes in the GTDB. For the “Ca. Methanoperedens”-associated Borg sequences, 50 kamA and 37 ablB gene-encoding amino acid sequences were manually retrieved from the original publication(Al-Shayeb et al., 2022). Both genes, kamA and ablB, were categorized according to their taxonomy, with a particular focus on archaeal subgroups (Fig. 3AB and Supplementary Table 3 and 4). 6
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