Reva, Oleg N.La Cono, ViolettaCrisafi, FrancescaSmedile, FrancescoMudaliyar, ManasiGhosal, DebnathGiuliano, LauraKrupovic, MartYakimov, Michail M.2024-05-172024-05-172024-04Reva, O.N., La Cono, V., Crisafi, F., Smedile, F., Mudaliyar, M., Ghosal, D. et al. (2024) Interplay of intracellular and trans-cellular DNA methylation in natural archaeal consortia. Environmental Microbiology Reports, 16(2), e13258. Available from: https://doi.org/10.1111/1758-2229.13258.1758-2229 (online)10.1111/1758-2229.13258http://hdl.handle.net/2263/96035DATA AVAILABILITY STATEMENT : In-house software tool SeqWord MotifMapper 3.1 developed for this study for visualization and analysis of methylation profiles supplemented with all input files: GBK genomes and GFF files generated by the program ipdSummary for H. lucertense and its symbiont, containing genome methylation data at different growth conditions: https://zenodo.org/doi/10.5281/zenodo.10700787. Accession numbers in the NCBI repository: Whole genome sequences: Halorhabdus sp. SVX81 – CP104322; H. lucertense SVX82 – CP104741-44; and Ca. Na. occultus SVXNc – CP104395; PacBio raw read data: Archaeal consortium I, PacBio Exp 1.1 – SRX22141242; Archaeal consortium I, PacBio Exp 1.2 – SRX22141245; Archaeal consortium II, PacBio Exp 2.1 – SRX22141234; Archaeal consortium II, PacBio Exp 2.2 – SRX22141235; Archaeal consortium III, PacBio Exp 3.1 – SRX22141243; Archaeal consortium III, PacBio Exp 3.2 – SRX22141244; Archaeal consortium IV, PacBio Exp 4 – SRX22141246; RNA-seq data: Archaeal consortium I, Illumina-RNA Exp 1.1 – SRX22141247; Archaeal consortium I, Illumina-RNA Exp 1.2 – SRX22141248; Archaeal consortium II, Illumina-RNA Exp 2.1 – SRX22141249; Archaeal consortium I, Illumina-RNA Exp 2.2 – SRX22141236; Archaeal consortium III, Illumina-RNA Exp 3.1 – SRX22141237; Archaeal consortium III, Illumina-RNA Exp 3.2 – SRX22141238; Archaeal consortium IV; Archaeal consortium IV, Illumina-RNA Exp 4.1 – SRX22141239; Archaeal consortium IV, Illumina-RNA Exp 4.2 – SRX22141240; Illumina-RNA Exp 4.3 – SRX22141241; PacBio methylation data: H. lucentense Exp.I – SUPPF_0000005533; H. lucentense Exp.II – SUPPF_0000005534; H. lucentense Exp.III – SUPPF_0000005535; H. lucentense Exp.IV – SUPPF_0000005536; Ca. Nanohaloarchaeota archaeon SVXNc Exp.II – SUPPF_0000005537; Ca. Nanohaloarchaeota archaeon SVXNc Exp.IV – SUPPF_0000005538.SUPPLEMENTARY FIGURE S1. Atlas view of the genome of H. lucertense SVX82g composed of the chromosome and the three plasmids shown as brown and dark-green arcs. Genomic islands identified by the SeqWord Genome Island Sniffer and the respective metrics: GC content; the ratio of generalized relative variance (GRV) versus relative variance (RV) of distribution of nucleotide tetramers normalized by GC content (n1_4mer); and distance D between local and global tetramer frequency patterns calculated in a 5 kbp sliding window stepping 2 kbp used for detection of genomic islands (see http://seqword.bi.up.ac.za/sniffer/index.html for more detail) are shown respectively by pink blocks and coloured histograms as explained in the legend. Locations of methyltransferase (MT) and restriction endonuclease (RE) genes are depicted by triangle red marks.SUPPLEMENTARY FIGURE S2. Venn diagrams, contingency tables, and estimated Chi2 metrics visualize the distribution of unmethylated CTAG motifs on the chromosome (A) and the plasmids (B); and unmethylated CCTTG motifs on the chromosome (C) and the plasmids (D) of H. lucertense SVX82 in different experiments: (I) pure (axenic) culture on d-xylose; (II) binary culture with the ectosymbiont Ca. N. occultus SVXNc on d-xylose; (III) binary culture with Halorabdus sp. SVX81 on xylan; (IV) trinary culture with Halorabdus sp. SVX81 and the ectosymbiont Ca. N. occultus SVXNc on xylan.SUPPLEMENTARY FIGURE S3. Venn diagrams, contingency tables, and estimated Chi2 metrics visualize the distribution of unmethylated GTCGAGG motifs on the chromosome (A) and the plasmids (B); and unmethylated GRAGAAG motifs on the chromosome (C) and the plasmids (D) of H. lucertense SVX82 in different experiments: (I) pure (axenic) culture on d-xylose; (II) binary culture with the ectosymbiont Ca. N. occultus SVXNc on d-xylose; (III) binary culture with Halorabdus sp. SVX81 on xylan; (IV) trinary culture with Halorabdus sp. SVX81 and the ectosymbiont Ca. N. occultus SVXNc on xylan.SUPPLEMENTARY FIGURE S4. Venn diagrams, contingency tables, and estimated Chi2 metrics visualize the distribution of methylated GDGcHC motifs on the chromosome (A) and the plasmids (B) of H. lucertense SVX82 in different experiments: (I) pure (axenic) culture on d-xylose; (II) binary culture with the ectosymbiont Ca. N. occultus SVXNc on d-xylose; (III) binary culture with Halorabdus sp. SVX81 on xylan; (IV) trinary culture with Halorabdus sp. SVX81 and the ectosymbiont Ca. N. occultus SVXNc on xylan.SUPPLEMENTARY TABLE S1. Composition of consortia used in this study (120 h of cultivation).SUPPLEMENTARY TABLE S2. SRA NCBI database accession numbers of the raw SMRT PacBio genomic reads and Illumina RNA reads generated for this study.SUPPLEMENTARY TABLE S3. Protein coding genes H. lucertense SVX82 with cTAG methylation within 20 bp upstream of the start codon.SUPPLEMENTARY TABLE S4. Methylation and expression of genes with multiple GRAGa G methylation motifs within their sequences.SUPPLEMENTARY VIDEO S1. Three-dimensional (segmented) view of host (SVX82, maroon outer layer) and DPANN (SVXNc, cyan outer layer) interaction showing extensive membrane blebbing from the DPANN SVXNc at the DPANN-host interface.DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano-sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction-modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self-restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo-ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.en© 2024 The Authors. Environmental Microbiology Reports published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License.DNA methylationArchaeal methylomicsTripartite xylanolytic halophilic consortiumSDG-15: Life on landArticle