LIQUID WATER IS ESSENTIAL TO LIFE ON EARTH; HOWEVER MOST PLANETS AND MOONS IN OUR SOLAR SYSTEM HAVE SURFACE TEMPERATURES WELL BELOW THE FREEZING POINT OF PURE WATER. EUROPA ENCELADUS CERES TITAN AND OTHER ICY BODIES ARE TARGETS FOR ASTROBIOLOGICAL INVESTIGATION. MARS IS PRESENTLY COLD AND DRY BUT ORBITAL OBSERVATIONS HAVE IDENTIFIED FLOWING LIQUID WATER AND THERE IS ABUNDANT EVIDENCE FOR PAST HABITABLE ENVIRONMENTS. EARTH MAY ALSO HAVE UNDERGONE A SERIES OF GLOBAL GLACIATIONS (SNOWBALL EARTH EVENTS) IN ITS EARLY HISTORY. THE ABUNDANCE OF ICY CONDITIONS IN OUR SOLAR SYSTEM SUGGESTS THAT LIFE IN FROZEN ENVIRONMENTS MAY PROVIDE ANSWERS TO QUESTIONS ABOUT THE ORIGIN EVOLUTION AND ULTIMATE FATE OF MICROBIAL CELLS AND THEIR BIOSIGNATURES. BACTERIA THAT ARE GROWING METABOLICALLY ACTIVE AND/OR SURVIVING IN THE PRESENCE OF ICE MAY HAVE CHARACTERISTICS THAT REFLECT THE EVOLUTION AND NATURE OF PRIMITIVE LIFE. THESE BACTERIA ARE INTERESTING TO ASTROBIOLOGY POLAR ECOLOGY AND CRYOPRESERVATION BUT THEIR ABILITY TO FUNCTION AT TEMPERATURES BELOW -5 DEG C IS POORLY UNDERSTOOD. WE HAVE RECENTLY SHOWN THAT THE MARINE PSYCHROPHILE COLWELLIA PSYCHRERYTHRAEA STR. 34H [CP34H] HAS DISTINCT SHIFTS IN PROTEIN EXPRESSION AND METABOLIC ACTIVITY AT -10 DEG C USING PROTEOMIC MASS SPECTROMETRY (NUNN ET AL. 2015). PRELIMINARY WORK IN OUR LABORATORIES REVEAL THAT THIS ORGANISM IS CAPABLE OF GROWING IN NUTRIENT BROTH WITH MARS ANALOG BRINE CONTAINING PERCHLORATE. THE OBJECTIVES OF THE PROPOSED RESEARCH ARE TO: 1) DEVISE THREE LOW-TEMPERATURE LIQUID-WATER ENVIRONMENTS THAT MIMIC THE KNOWN CHEMISTRY OF BRINES: I) IN SEA ICE ON MODERN EARTH II) ON SNOWBALL EARTH AND III) ON MARS; 2) MEASURE MICROBIAL GROWTH RATE METABOLIC ACTIVITY ABILITY TO SURVIVE WHILE INACTIVE AND LONGEVITY FOR PSYCHROPHILES IN THESE THREE ENVIRONMENTS; 3) REVEAL PROTEOMIC BIOSIGNATURES FOR GROWTH ACTIVITY AND SURVIVAL STRATEGIES AND UNDERSTAND KEY MOLECULAR RESPONSES OF LIFE IN THESE THREE ENVIRONMENTS. METHODOLOGY: WE PROPOSE A 3-YEAR PROJECT TO PERFORM NOVEL PROTEOMIC EXAMINATIONS ON A SUITE OF THREE PSYCHROPHILES (CP34H PSYCHROBACTER SP. STR. 7E AND A MODEL HALOPHILE) HELD FOR 12 MONTHS AT SUBZERO TEMPERATURES (-1 -5 -10 -30 -50 AND -70 DEG C; REPRESENTATIVE OF ICE ABOVE AND BELOW THE EUTECTIC OF SEAWATER AND PUTATIVE MARS BRINES) IN COMPARISON TO OPTIMAL GROWTH TEMPERATURE BENCHMARK DATA (8 DEG C). BACTERIA WILL BE INTRODUCED TO SAMPLES REPRESENTATIVE OF MODERN SEA ICE COLD SEA ICE HYPOTHESIZED TO OCCUR ON SNOWBALL EARTH AND ICE ON MARS. WE WILL SPIKE CULTURES WITH 3H-LEUCINE 3H-THYMIDINE OR 13C-LEUCINE INCUBATED AT SPECIFIED TEMPERATURES AND SAMPLE THROUGH TIME TO TRACK METABOLIC ACTIVITIES VIABILITY CELL DEATH AND PROTEOME CHANGES. WE EXPECT THAT NOVEL 13C-LEUCINE TRACKING AND GLOBAL PROTEOME RESPONSE QUANTIFICATIONS USING MASS SPECTROMETRY WILL ALLOW FOR THE IDENTIFICATION AND MONITORING OF INDIVIDUAL NEWLY SYNTHESIZED PROTEINS AND WILL PROVIDE DOMINANT BIOMARKERS FOR GROWTH AND/OR SURVIVAL. THIS RESEARCH HAS VALUE TO UPCOMING SPACE-EXPLORATION LIFE DETECTION MISSIONS. IDENTIFICATION OF PROTEINS NEWLY SYNTHESIZED IN LOWT EMPERATURE ENVIRONMENTS OR PROTEINS INDICATIVE OF LONG-TERM ICE SURVIVAL WILL PROVIDE BIOSIGNATURES TO TARGET WHEN EXPLORING LIFE IN LOW-TEMPERATURE ECOSYSTEMS RELEVANT TO FUTURE EXPLORATION. IN ADDITION UNDERSTANDING KEY METABOLIC STRATEGIES FOR LONG-TERM SURVIVAL IN ICE WILL PROVIDE CLUES ON EARLY EVOLUTION AND SURVIVAL OF LIFE AS EARTH UNDERWENT EXTENSIVE GLACIATION DURING THE NEOPROTEROZOIC ERA. THIS PROPOSAL DIRECTLY ADDRESSES THE GOALS OF ROSES 2017 AND THE NASA EXOBIOLOGY PROGRAM SOLICITATION TO ADDRESS "THE POTENTIAL FOR THE ORIGIN AND ESTABLISHMENT OF LIFE UNDER CONDITIONS PREVAILING ON OTHER PLANETARY BODIES AND BASIC RESEARCH ON THE FORMATION AND RETENTION OF BIOSIGNATURES UNDER NON-EARTH CONDITIONS."