196 Chapter 6 only considering “Ca. Methanoperedens” transcripts expressed under 0% and 1.5% salinities (pink). Heatmap legend to the middle right of the panel refers to normalized “Ca. Methanoperedens” TPM (from 0 to 700) whereas the one below depicts metagenomic-based overall community normalized gene coverage (0.0001 to 0.2). DISCUSSION Our present study demonstrates that “Ca. Methanoperedens” acclimates to marine salinities (3%) and synthesizes N(ε)-acetyl-β-L-lysine during 12 weeks of salt increase (from 0 to 1.5%). This finding represents the description of a relevant osmolyte in the ANME group, achieved through an untargeted metabolomics approach. Our study demonstrates the power of untargeted metabolomics to discover uncharted metabolites in archaea. This osmolyte was first described in the methanogenic archaeon M. mazei (Sowers et al., 1990), although its encoding genes, kamA and ablB, were later discovered on the chromosomes of several methanogenic archaea(Pflüger et al., 2003). N(ε)-acetyl-β-L-lysine was also found in halophilic bacteria(Jiang et al., 2015; Joghee & Jayaraman, 2014) and was reported as a key excretion osmolyte upon hyposalinity stress in anaerobic granular sludgeassociated microbial consortia (Sudmalis et al., 2018b). Gene organization analysis of the N(ε)-acetyl-β-L-lysine key genes kamA and ablB encoding for its biosynthetic pathway shows that they are encoded in the same operon with an additional gene encoding for a poorly described medium-sized small conductance mechanosensitive channel (MscS)-like membrane protein: ynaI (Flegler et al., 2020). So far, the homologous MscS protein from Escherichia coli is the best characterized mechanosensitive channel from this protein family and it protects Eschericia coli during hypoosmotic shock (Levina et al., 1999; Malcolm & Maurer, 2012; Vásquez et al., 2008). Although there is no direct physiological evidence on the role of YnaI yet (Wilson et al., 2013), our data indicates an upregulation of ynaI under increased salinities. Our metagenome indicated that ablB is only found in “Ca. Methanoperedens” in the here investigated culture and thus directly linking N(ε)-acetyl-β-L-lysine to ANME in the enrichment. Only low expression of kamA and ablB was found
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