Methylation of DNA in Bacteria has been reported to play roles in DNA replication, restriction/modification systems, and protein expression. We recently demonstrated that DNA is an excellent source of phosphorus, a limited source for nitrogen and not at all a carbon source for growing the archaeal hypersaline adapted species Haloferax volcanii. Furthermore, we also determined that H. volcanii (and all other halobacterial species examined) grows better (or not at all) on some sources of DNA than others, and that this bias is caused by methylation of the DNA. This previously unknown role for DNA methylation has important implications for the evolution of species, and the cycling of nutrients in hypersaline environments. Our goal is to investigate how methylation affects DNA uptake, and nutrient cycling in NaCl saturated brines. The main objectives of the research are: 1)Determine the motifs of methylation sites for all seven of the putative methyltransferase genes in H. volcanii. 2)Compare growth of H. volcanii on DNA from all H. volcanii methyltransferase mutants, and in different combinations. Compare growth of H. volcanii on different species DNA, including different strains from the same species that have different methyltransferases. 3)Extract environmental cellular-bound DNA, sequence the metagenome. Use the same DNA as a nutrient to grow Haloferax volcanii, Halorubrum sp., Halobacterium sp. and Haloarcula sp. Extract the unutilized DNA and sequence it. Compare the pre and post DNA consumption states for biases in uptake. 4)Extract environmental cell-free (water column) and cell-bound DNA from natural saturated brines. Sequence the metagenome of each. Add C, N, and P to microcosm experiments, extract the cell-free and cell-bound DNA, and compare to initial condition. Additionally, measure natural C, N, and P concentrations prior to and after amending microcosms. Summary of methodology We will primarily use DNA sequencing to determine the methylome of H. volcanii, for analysis of growth experiments, and for the microcosm metagenomic analyses. Bioinformatic analyses of genomic and metagenomic data will be performed on the sequence data to determine methylation motifs and for analysis of pre and post DNA utilization community composition. Relevance The goal of research funded by the Exobiology program is to understand the physiology of microorganisms including extremophiles; to investigate key biological processes and their environmental impact; study the coevolution of microbial communities, and interactions within such communities, that drive major geochemical cycles. The work proposed would address these goals by providing insight into DNA metabolism of extreme salinity adapted Archaea, and how these hypersaline adapted Archaea contribute to the phosphorus cycle.