Description of key central objectives: Sulfur assimilation and trafficking in methanogens exhibit unique features as compared with all other extant organisms, reflecting the emergence of these Archaea on the anaerobic Earth over two billion years ago. Perhaps the most essential distinguishing characteristic is the uptake of sulfur in its highly reduced sulfide form. Unlike almost all other organisms on the contemporary Earth, many methanogens lack recognizable genes specifying proteins involved in sulfate assimilation as well as synthesis of cysteine, iron-sulfur clusters, and other key sulfur-containing cellular metabolites including RNAs. The central objective of the proposal is to identify novel genes involved in sulfide uptake and sulfur trafficking, and to characterize the protein products of these genes and their interaction partners in the cell. Concise statement of methods: To accomplish these objectives we will apply a variety of in silico and experimental tools. Using exhaustive bioinformatics analysis applied to all known archaeal genomes, we are identifying previously uncharacterized candidate genes for sulfide uptake and sulfur trafficking. An in vivo approach using single and multiple chromosomal knockouts in the organism Methanosarcina acetivorans will then be used to elucidate specific sulfur-related phenotypes in growing cells. We will then investigate function more directly by a variety of biochemical approaches, including endogenous expression of tagged proteins, quantitative affinity purification, and mass spectrometry. Perceived significance of the proposed work: These experiments are highly relevant to this call and to the NASA Astrobiology Roadmap, particularly with respect to understanding how life on Earth and its planetary environment have co-evolved through geologic time. A second theme is to achieve deeper understanding of the evolutionary mechanisms and environmental limits of life. By discovering novel sulfur metabolic genes present in still-extant microorganisms that originated as long as 3.5 billion years ago, we will be positioned to use phylogenetic analysis to trace evolutionary histories of the key proteins. Molecular adaptations necessary to bridge from the anaerobic to the aerobic Earth may then also become clear in comparing genes and proteins from organisms that differentiated at particular junctures. Finally, this work will also inform studies of how the global biogeochemical sulfur cycle has changed through geologic time with the emergence of the new pathways that are present in most organisms today.