MICROBES ARE THE MOST ABUNDANT MOST DIVERSE AND OLDEST FORMS OF LIFE ON EARTH THE ONLY PLANET IN THE UNIVERSE THAT WE KNOW SUPPORTS LIFE. AS SUCH MICROBIAL LIFE ON EARTH IS THE BEST SOURCE OF INFORMATION AVAILABLE TO US FOR PREDICTING THE HABITABILITY OF OTHER WORLDS AND FOR DETERMINING WHICH POTENTIAL BIOSIGNATURES ARE MOST LIKELY TO RESULT IN THE DETECTION OF LIFE ELSEWHERE. OUR WORK WILL CONTINUE TO CHARACTERIZE MICROBIAL LIFE ON EARTH IN RELEVANT ANALOG ENVIRONMENTS IN ORDER TO UNDERSTAND THE ORIGIN AND EVOLUTION OF MICROBIAL LIFE ON EARTH AND TO INFORM THE SEARCH FOR LIFE ELSEWHERE. WE WILL FOCUS SPECIAL ATTENTION ON THE PROCESS OF EVOLUTION WHICH HAS NOT ONLY PRODUCED THE ASTOUNDING MICROBIAL DIVERSITY WE OBSERVE BUT IS DIRECTLY RESPONSIBLE FOR THE PRODUCTION OF MOST OF THE POTENTIAL BIOSIGNATURES CURRENTLY TARGETED BY UPCOMING MISSIONS. OUR TEAM S COORDINATED STUDY OF THE EVOLUTIONARY RECORD OF EXTANT LIFE WILL INFORM THE MECHANISM HISTORY AND DISTRIBUTION OF BIOSIGNATURES LEAD TO THE IDENTIFICATION OF KEY PROCESSES THAT HAVE EXPANDED HABITABILITY (OR ALLOW LIFE AT THE LIMITS OF HABITABILITY) AND IDENTIFY FEATURES AMENDABLE TO LIFE DETECTION EFFORTS. WE WILL CONDUCT MANIPULATIONS OF INTACT MICROBIAL MATS AND MATS CONSTRUCTED USING CULTURES TO INVESTIGATE THE RESPONSE OF THE MATS TO ENVIRONMENTAL FORCING FUNCTIONS (E.G. LIGHT TEMPERATURE AND FLOW). WE WILL UTILIZE STATE-OF-THE-ART MOLECULAR ECOLOGICAL TECHNIQUES IN ORDER TO FOLLOW CHANGES IN MICROBIAL COMMUNITY COMPOSITION IN THE MATS. WE WILL ALSO CONDUCT A COMPREHENSIVE EXAMINATION AND QUANTIFICATION OF THE N CYCLE IN PHOTOSYNTHETIC MICROBIAL MATS BY INTEGRATING BIOGEOCHEMICAL MEASUREMENTS INCLUDING STABLE-ISOTOPE TRACER EXPERIMENTS AND A MULTIFACETED MOLECULAR BIOLOGY APPROACH INCLUDING METAGENOMICS METATRANSCRIPTOMICS AND TARGETED AMPLICON SEQUENCING. THESE METHODS WILL BE APPLIED ACROSS A COMBINATION OF BOTH FRESHLY COLLECTED MATS AND MATS SUBJECTED TO EXPERIMENTAL MANIPULATIONS REPRESENTING CURRENT AND ANCIENT-EARTH ENVIRONMENTAL CONDITIONS. WE WILL BE SAMPLING FROM BOTH THE OXIC FIRST FEW MILLIMETERS AT THE MAT S SURFACE AS WELL AS DEEPER IN THE MAT (FROM THE ANOXIC PORTION). BIOGEOCHEMICAL MEASUREMENTS AND STABLE-ISOTOPE TRACER TECHNIQUES WILL RESULT IN THE COMPLETE QUANTIFICATION OF THE N CYCLE (SOURCES SINKS AND INTERNAL TRANSFORMATIONS). THE PROPOSED MOLECULAR TECHNIQUES WILL ALLOW INVESTIGATION OF THE MICROBIAL COMMUNITIES AND THE GENE EXPRESSION PATTERNS RESPONSIBLE FOR THESE TRANSFORMATIONS AS WELL AS PHYLOGENETIC ANALYSIS OF THE N-CYCLING GENES RECOVERED FROM EACH SYSTEM BY TARGETED AMPLICON SEQUENCING AND PHYLOGENOMIC ANALYSES OF NOVEL REPRESENTATIVE GENOMES. IN A RELATED BUT SEPARATE TASK WE WILL PERFORM COMPARATIVE ANALYSIS OF EXISTING GENOMIC DATA WITHIN IN A QUANTITATIVE PHYLOGENOMIC FRAMEWORK COUPLED WITH INFORMED CULTUREBASED VALIDATION TO IDENTIFY WHEN AND HOW THE EVOLUTION OF KEY TRAITS AND SPECIES OCCURRED.