Genomic insights into the Archaea inhabiting an Australian radioactive legacy site

<strong>Abstract</strong> During the 1960s, small quantities of radioactive materials were co-disposed with chemical waste at the Little Forest Legacy Site (LFLS, Sydney, Australia). The microbial function and population dynamics during a rainfall event using shotgun metagenomics has been previously investigated. This revealed a broad abundance of candidate and potentially undescribed taxa in this iron-rich, radionuclide-contaminated environment. Here, applying genome-based metagenomic methods, we recovered 37 refined archaeal bins (≥50% completeness, ≤10% redundancy) from 10 different major lineages. They were mostly included in 4 proposed lineages within the DPANN supergroup (LFWA-I to IV) and <em>Methanoperedenaceae</em>. The new <em>Methanoperedens</em> spp. bins, together with previously published data, suggests a potentially widespread ability to use nitrate (or nitrite) and metal ions as electron acceptors during the anaerobic oxidation of methane by <em>Methanoperedens</em> spp. While most of the new DPANN lineages show reduced genomes with limited central metabolism typical of other DPANN, the candidate species from the proposed LFWA-III lineage show some unusual features not often present in DPANN genomes, i.e. a more comprehensive central metabolism and anabolic capabilities.While there is still some uncertainty about the capabilities of LFW-121_3 and closely related archaea for the biosynthesis of nucleotides <em>de novo</em>, and amino acids, it is to date the most promising candidate to be the first <em>bona fide</em> free-living DPANN archaeon. <strong>Repository Contents</strong> <em>genomes.tar:</em> includes each of the reference and novel assembled MAG/bins used for analysis in the main manuscript as .tar.gz compressed folders. Each genome folder contains the output from: Anvi'o, EggNOG analysis with arNOG library, InterProScan analysis, rRNA search with Barrnap, output from searching high heme cytochromes (≥10 heme binding sites in a single protein), CAZy search output, MEROPS output (BLASTp), PSORTb, and TCDB. In the case of the new MAGs (i.e. LFW_Bin_00*, referred in the paper as LFW-*), some additional contents are included: tRNAs from tRNAscan-SE, and Prokka annotation files. <em>pangenomics.tar</em>: includes the Anvi'o files for the pangenomic analysis of: <em>anme-2d.tar.gz</em>: pangenome of <em>Methanoperedens</em> spp. based on 4 reference and 6 novel MAGs clustered with an MCL inflation value of 6.0. It also includes the output of ANI analysis (via pyANI) and AAI (via CompareM). <em>LFWA-III.tar.gz</em>: pangenome analysis of the LFWA-III lineage ('Gugararchaeaceae) based on 2 reference and 8 novel MAGs clustered at inflation values of 1.0, 1.5 and 2.0. It also includes the output of ANI analysis (via pyANI) and AAI (via CompareM). <em>phylogeny.tar</em>: includes the files required for the phylogenomic/phylogenetic analyses shown in the paper and the Supplementary Information: <em>rp44.tar.gz</em>: phylogenomic analysis based on 44 universal and archaeal-specific ribosomal proteins (Figure 1 in paper). It also includes annotation files for iTOL. <em>NarG.tar.gz</em>: phylogeny of NarG and relate molybdopterin oxidoreductase proteins (Figure 5 in paper). <em>LysJ-ArgD.tar.gz</em>: phylogeny of LysJ/ArgD proteins and their orthologous (Figure S3 in paper).