175 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress The libraries were pooled, denatured and sequenced with the MiSeq (Illumina) sequencer (San Diego, California USA). Paired end sequencing of 2x300bp was performed using the MiSeq Reagent Kit v3 (San Diego, California USA) according to the manufacturer’s protocol yielding 4,584,058 reads. Biogeography on “Candidatus Methanoperedens Strain Vercelli 1” The biogeography of “Ca. Methanoperedens Vercelli Strain 1”, identified by GTDBTk v2.1.0 as s__905339155.1, was assessed by querying operational taxonomic units (OTUs) categorized under the same species. This search utilized keywords for species numbers and was conducted across publicly available metagenomes using Sandpiper (July 2022) (https://sandpiper.qut.edu.au/) (see Supplementary Table 2). Gene based metagenomic analysis Softwares Artemis v16.0.0 and Operon-mapper (https://biocomputo.ibt.unam.mx/ operon_mapper/) (accessed on August 16, 2023) were used for a comprehensive gene visualization and prediction of the operon conforming the osmolyte production genes of interest, respectively(Carver et al., 2012; Taboada et al., 2018). Genes encoding sialic acid biosynthesis were obtained via BLASTp from reference pathways of Campylobacter jejunii (Schoenhofen et al., 2009) and Halorubrum sp. PV6 (Zaretsky et al., 2018) with e-value thresholds below e^-20 including a protein identity threshold of >30%. To retrieve a read and contig size corrected gene coverage, we employed CoverM with the -contig flag, with a minimum identity of 95% and a minimum aligned read length of 75% for each read (https://github. com/wwood/CoverM). We also corroborated the annotation of genes involved in sialic acid biosynthesis with HMM profiles building PFAM annotations of genes of interest, with a hmm alignment threshold of 10e^-5. When possible, HMMs that were stringently KEGG-curated (K15897/pseG and K15912/legB) with pre-defined adaptive thresholds of kofamKOALA (https://www.genome.jp/tools/kofamkoala/) (accessed on September 2023) (Aramaki et al., 2020). Both for PFAM and HMM annotations, relative abundances were calculated based on summed coverage per metagenome, either freshwater or brackish. 6
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