SCIENTIFIC GOALS AND OBJECTIVES A CENTRAL GOAL OF OUR RESEARCH IS ACHIEVING A DEEPER UNDERSTANDING OF THE UNUSUAL SULFIDE-BASED ASSIMILATORY SULFUR METABOLISM THAT IS FOUND AMONG EARTHS MOST PRIMITIVE ANAEROBIC ORGANISMS. WE HAVE DISCOVERED FOUR NEW GENES THAT ARE CONSERVED AMONG METHANOGENS IN HIGHLY SULFIDIC HABITATS AND HAVE ELUCIDATED THE PHYSIOLOGICAL OR BIOCHEMICAL ROLES OF SEVERAL OF THE ENCODED PROTEINS. OUR NEXT OBJECTIVES INVOLVE MORE INTENSIVE STRUCTURE-FUNCTION ANALYSIS AND INVESTIGATION OF HOW ENVIRONMENTAL SULFIDE IS FURTHER MOBILIZED FOR INCORPORATION INTO KEY CELLULAR METABOLITES RNAS AND PROTEINS. THE SECOND MAJOR GOAL OF THE RESEARCH IS TO UNDERSTAND HOW THESE MICROBIAL SULFUR ASSIMILATION PROCESSES ON THE ANCIENT EARTH EVOLVED IN CONCERT WITH CHANGES IN THE GLOBAL BIOGEOCHEMICAL ENVIRONMENT. WE FOCUS ESPECIALLY ON THE SHARP INCREASE OF OXYGEN CONCENTRATIONS IN THE OCEANS AND ATMOSPHERE THAT OCCURRED FOLLOWING THE ADVENT OF OXYGENIC PHOTOSYNTHESIS TWO TO THREE BILLION YEARS AGO. APPROACH AND METHODOLOGY THE OBJECTIVES WILL BE ADDRESSED USING BOTH EXPERIMENTAL AND COMPUTATIONAL TOOLS. BIOCHEMICAL APPROACHES TO CHARACTERIZING METHANOGEN PROTEINS INVOLVED IN SULFIDE UPTAKE AND ASSIMILATION INCLUDE X-RAY CRYSTALLOGRAPHY AND ENZYME KINETICS. THESE METHODS WILL BE COMPLEMENTED BY OTHER TECHNIQUES FOR TRACKING PROTEIN-PROTEIN INTERACTIONS IN CELLS INCLUDING ENDOGENOUS EXPRESSION OF TAGGED PROTEINS QUANTITATIVE AFFINITY PURIFICATION AND MASS SPECTROMETRY. TO INVESTIGATE HOW ANAEROBIC SULFIDE ASSIMILATION PROCESSES EVOLVED UPON THE ADVENT OF MOLECULAR OXYGEN ON THE EARLY EARTH WE WILL EMPLOY METHODS DERIVED FROM SYNTHETIC BIOLOGY TO RECONSTRUCT THE ANAEROBIC SULFUR ASSIMILATION APPARATUS IN THE COMMON BACTERIUM ESCHERICHIA COLI WHICH IS ABLE TO GROW BOTH AEROBICALLY AND ANAEROBICALLY. RELEVANCE TO THE EXOBIOLOGY PROGRAM ELEMENT 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 CHARACTERIZING NOVEL SULFUR METABOLIC PROTEINS PRESENT IN STILL-EXTANT MICROORGANISMS THAT ORIGINATED AS LONG AS 3.5 BILLION YEARS AGO WE WILL BE ABLE TO TRACE EVOLUTIONARY HISTORIES OF THE KEY PROTEINS. MOLECULAR ADAPTATIONS NECESSARY TO BRIDGE FROM THE ANAEROBIC TO THE AEROBIC EARTH SHOULD BECOME CLEAR IN COMPARING GENES AND PROTEINS FROM ORGANISMS THAT DIFFERENTIATED AT PARTICULAR JUNCTURES. FINALLY THIS WORK WILL ALSO INFORM STUDIES OF HOW ANCIENT CELLS ADAPTED TO THE INCREASE IN OCEANIC AND ATMOSPHERIC OXYGEN CONCENTRATIONS TRIGGERING THE EMERGENCE OF THE NEW SULFUR ACQUISITION PATHWAYS THAT ARE PRESENT IN MOST ORGANISMS TODAY.