SCIENTIFIC GOALS AND OBJECTIVES. THIS PROPOSAL WILL ADDRESS CORE GOALS OF NASA'S EXOBIOLOGY PROGRAM THROUGH RESEARCH THAT EMPHASIZES A NOVEL MICROBIAL PROCESS- CARBON MONOXIDE (CO) OXIDATION UNDER OXIC SUB-OXIC AND ANOXIC CONDITIONS BY MEMBERS OF THE EURYARCHAEOTA INCLUDING EXTREME HALOPHILES AND ARCHAEOGLOBUS FULGIDUS. THE MAJOR RESEARCH GOALS INCLUDE: (1) DETERMINE THE CAPACITY OF EURYARCHAEAL CO OXIDIZERS TO USE LEVELS OF CO PRESENT IN TERRESTRIAL AND EXTRATERRESTRIAL ATMOSPHERES AS SOURCES OF ENERGY FOR SURVIVAL OR GROWTH AND AS A FUNCTION OF VARIOUS ELECTRON ACCEPTORS AND CRITICAL ENVIRONMENTAL VARIABLES (E.G. WATER POTENTIAL); (2) DETERMINE THE DIVERSITY OF EURYARCHAEAL CO OXIDIZERS AND CONTROLS OF DIVERSITY IN GEOCHEMICALLY DISTINCT HYPERSALINE SYSTEMS; (3) DEVELOP A MODEL FOR THE ORIGIN AND EVOLUTION OF MOLYBDENUM-DEPENDENT CARBON MONOXIDE DEHYDROGENASE THE KEY ENZYME USED BY CO-OXIDIZING HALOARCHAEA. SEVERAL SPECIFIC OBJECTIVES WILL BE PURSUED IN ORDER TO ACCOMPLISH THE RESEARCH GOALS: OBJECTIVE 1. ASSESS THE CAPACITY OF A. FULGIDUS TO USE CO FOR GROWTH AND MAINTENANCE AS A FUNCTION OF CO CONCENTRATION AND ELECTRON ACCEPTOR AVAILABILITY INCLUDING USE OF (PER)CHLORATE. OBJECTIVE 2. DETERMINE THE CAPACITY OF TYPE 2 CO-OXIDIZING HALOARCHAEA TO USE ATMOSPHERIC CO AS AN ENERGY SOURCE FOR SURVIVAL UNDER CONDITIONS RELEVANT FOR MARS AND OTHER SYSTEMS. OBJECTIVE 3. ASSESS THE PHYLOGENETIC DIVERSITY OF TYPE 2 CO-OXIDIZING HALOARCHAEA IN HYPERSALINE SYSTEMS AND POTENTIAL CONTROLS OF THEIR DIVERSITY. OBJECTIVE 4. DEVELOP A DATABASE OF NEW TYPE 2 CO-OXIDIZING HALOARCHAEAL MO-CODH GENES TO ASSESS THEIR ORIGIN AND EVOLUTIONARY HISTORY THROUGH PHYLOGENETIC ANALYSES. BRIEF SUMMARY OF METHODS. NEW CO-OXIDIZING HALOARCHAEAL ISOLATES WILL BE OBTAINED FROM FIELD SITES WHERE CO UPTAKE HAS BEEN DOCUMENTED USING IN SITU FIELD ASSAYS AND EX SITU ANALYSES. NEW AND EXISTING ISOLATES WILL BE USED TO ASSESS CAPACITIES FOR CO OXIDATION UNDER A WIDE RANGE OF CONDITIONS THAT WILL ESTABLISH THE PHYSIOLOGICAL RANGE OF THE PROCESS AND POTENTIAL FOR ACTIVITY IN AN EXTRATERRESTRIAL CONTEXT. THESE ASSAYS WILL EMPHASIZE LOW CO LEVELS WATER STRESS VARIED ELECTRON ACCEPTORS AND PERSISTENCE. NEXTGEN SEQUENCING OF 16S RRNA GENES WILL BE USED TO DETERMINE THE DIVERSITY OF HALOARCHAEAL COMMUNITIES AS A WHOLE AND ANALYSES OF COXL GENES WILL BE USED TO ASSESS CO-OXIDIZING HALOARCHAEA. SEQUENCES OF CODH GENES AND VARIOUS HOUSEKEEPING GENES WILL BE SUBJECTED TO PHYLOGENETIC ANALYSES BASED ON MULTIPLE ALGORITHMS THE RESULTS OF WHICH WILL BE USED TO INFER THE EVOLUTIONARY HISTORY OF MO-DEPENDENT CODHS. PROPOSAL RELEVANCE. THE WORK PROPOSED HERE IS DIRECTLY RELEVANT FOR AT LEAST TWO AREAS OF EARLY EVOLUTION OF LIFE AND THE BIOSPHERE. FIRST ANTICIPATED RESULTS WILL PROVIDE NOVEL INSIGHTS INTO THE ECOPHYSIOLOGY AND DIVERSITY OF CO-OXIDIZING EURYARCHAEOTA WHOSE CHARACTERISTICS MAY REFLECT THE NATURE OF EARLY BIOTIC SYSTEMS ON EARTH MARS OR ELSEWHERE. SECOND THE RESEARCH WILL INVESTIGATE THE PHYLOGENETIC HISTORY OF AN ECOLOGICALLY AND BIOGEOCHEMICALLY IMPORTANT PROCESS CO OXIDATION AND ITS ENVIRONMENTAL IMPACT AS DETERMINED BY INTERACTIONS BETWEEN CO-OXIDIZING HALOPHILES AND OTHER HALOPHILIC FUNCTIONAL GROUPS.