189 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress LAM tree (black lines in panel A). Sequences were assigned to their lowest GTBD-Tk v2.1.0 taxonomical category or, clustered in clades and refer to their higher taxonomical categories or groups. Borg labels were remained unchanged from original publication. “Ca. Methanoperedens” ablB gene expression was classified depending on the salinity source employed on the different metatranscriptomic datasets. Annotated sequences included previously observed AT-encoding gene ablB expression (obtained from public metatranscriptomes) or, ablB-harboring “Ca. Methanoperedens” that are known to harbor a non-ablB carrying plasmid. Both kamA and ablB were widely distributed in bacteria and methane/alkane cycling archaea, both chromosomally and encoded on Borgs. Although most Borgs harbored both genes, a small fraction were only carrying kamA (Fig. 3 and Supplementary Fig. 10). The “Ca. Methanoperedens” kamA and ablB from this study (Fig. 3AB, pink star) clustered phylogenetically with those genes from other ANME archaea and were distantly related to the first reported reference in methanogenic archaea (Fig. 3AB, blue star). Furthermore, expression of the kamA and ablB genes closely related to those from our “Ca. Methanoperedens” have been reported in metatranscriptomic dataset from both brackish and freshwater origins (Fig. 3C and Supplementary Fig. 10). Both kamA and ablB genes were identified in MAGs representing the ANME-2a, 2c and 3, but not the 1 and 2b sub-groups. Although kamA and ablB genes from ANME groups clustered close to each other, the methanogenic kamA and ablB genes appeared to have a wider distribution that is intertwined with those encoded by bacteria (Fig. 3AB). Our data suggest potential horizontal gene transfer (HGT) of kamA and ablB from bacteria to ANME and halophilic methanogen Methanosalsum, with the “Ca. Methanoperedens” kamA and ablB genes being closely related to those from the bacterial phylum Firmicutes (Fig. 3C for ablB and Supplementary Fig. 10 for kamA). Metatranscriptome and metaproteome changes during salt stress In addition to the transcripts of kamA and ablB genes, a total of 286 “Ca. Methanoperedens” transcripts were upregulated at non-acclimated 1.5% salinity as compared to freshwater conditions, accompanied by 11 proteins being more abundantly present. Conversely, 264 “Ca. Methanoperedens” transcripts were downregulated, and 17 proteins were less abundantly present (threshold: P<0.05 difference between the 1.5% and 0% salinity) (Fig. 4 and Supplementary Fig. 11). Overall, the metaproteome seemed to exhibit a delayed response to the salt6
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