THE CURIOSITY ROVER HAS DETECTED BACKGROUND ATMOSPHERIC CH4 LEVELS OF 0.7 PPBV WITH SPIKES TO 7 PPBV [WEBSTER ET AL. 2015]. OF THE MANY MECHANISMS TO INJECT CH4 INTO THE MARTIAN ATMOSPHERE (E.G. SERPENTINIZATION UV DESTRUCTION OF ACCRETED ORGANICS ETC.) METHANOGENESIS IS THE ONLY BIOLOGICAL PROCESS PROPOSED [REID ET AL. 2006] THAT MIGHT BE ACTIVE IN THE SHALLOW SUBSURFACE ON MARS. ALSO LIQUID BRINES COMPOSED OF MG-PERCHLORATE MG-CHLORATE AND NA-PERCHLORATE HAVE BEEN DETECTED IN ACTIVE RECURRING SLOPE LINEAE (RSL) ON STEEP CRATER WALLS ON MARS [OJHA ET AL. 2015]. THE EXPERIMENTS OUTLINED HEREIN WILL ESTABLISH THE LOW-PRESSURE LOW-TEMPERATURE LOW-OXYGEN (PO2) AND HIGH PERCHLORATE AND CHLORATE SALT LIMITS FOR METABOLISM GROWTH AND CH4 PRODUCTION OF PSYCHROPHILIC METHANOGENS UNDER SIMULATED MARTIAN CONDITIONS. FURTHERMORE THE EXPERIMENTS WILL CHARACTERIZE THE TRANSCRIPTOMIC AND PROTEOMIC ALTERATIONS IN METHANOGENS WHEN GROWN UNDER CONDITIONS THAT MAY PERSIST IN SHALLOW SUBSURFACE RSL BRINES.THE RESEARCH WILL BE DIVIDED INTO TWO SECTIONS IN WHICH THE SCHUERGER LAB WILL FOCUS ON CHARACTERIZING THE LOW-PRESSURE LOWTEMPERATURE LOW-PO2 AND HIGH-SALT LIMITS OF GROWTH FOR PSYCHROPHILIC OR PSYCHROTOLERANT METHANOGENS FOR WHICH COMPLETE GENOMES EXIST (E.G. METHANOGENIUM FRIGIDIUM DSM 16458 METHANOCOCCOIDES BURTONII DSM 6242). THE ONSTOTT LAB WILL FOCUS ON CHARACTERIZING THE VARIABILITY OF THE TRANSCRIPTOMES AND PROTEOMES OF THE METHANOGENS AS FUNCTIONS OF EXPOSURE TO INCREASINGLY MARS-LIKE CONDITIONS TESTING THE LOW-PRESSURE LOW-TEMPERATURE LOW-PO2 AND HIGH PERCHLORATE/CHLORATE SALT LIMITS FOR METABOLISM AND GROWTH.A MARS SIMULATION CHAMBER (SCHUERGER LAB) WILL BE CONFIGURED WITH TWO INDEPENDENT MICROBIAL ASSAY CHAMBERS (MAC; UNITS ONHAND) IN WHICH THE BULK ATMOSPHERE OF EACH MAC UNIT CAN BE INDIVIDUALLY SAMPLED WITH A QUADRUPOLE MASS-SPEC RESIDUAL GAS ANALYZER (RGA; OPERATIONAL; MINIMUM DETECTION OF CH4 IS 0.5 PPM AT 0.7 KPA (7 MBAR)) OR GAS CHROMATOGRAPH (GC; MINIMUM DETECTION OF CH4 IS 3 PPM A 0.7 KPA). EXPERIMENTS WILL FOCUS ON THE METABOLIC RESPONSES OF THE METHANOGENS BETWEEN 5.0 KPA (THE CURRENT LOW PRESSURE GROWTH LIMIT FOR METHANOGENS [KRAL ET AL. 2011]) AND 0.7 KPA (MARTIAN SURFACE PRESSURE).WE WILL EXAMINE THE TEMPORAL EXPRESSION OF GENES USING RNA-SEQ AND PROTEOME ANALYSES USING LC-MS/MS AS THE METHANOGENS ARE SUBJECTED TO VARYING PRESSURE TEMPERATURE PO2 AND PERCHLORATE/CHLORATE SALT CONDITIONS. THE ASSEMBLED MRNA CONTIGS AND PROTEIN PEPTIDES WILL BE MAPPED TO THE HOST GENOMES AND THEIR COVERAGE AND ABUNDANCE MEASURED RESPECTIVELY. WE WILL USE A FLUORESCENT MOLECULAR BEACON TARGETING THE MCRA MRNA TO DETERMINE WHAT PROPORTION OF CELLS ARE ACTIVELY PRODUCING CH4 THEREBY PROVIDING A SPECIFIC CELLULAR CH4 PRODUCTION RATE UNDER THE DIVERSE CONDITIONS LISTED ABOVE. THE COMBINATION OF THESE TWO TOOLS WILL PROVIDE A COMPLETE PICTURE OF THE RESPONSE OF THE DIFFERENT METHANOGEN STRAINS TO DECREASING PRESSURE AND TEMPERATURE THE PRESENCE OF LOW PO2 AND TO ELEVATED PERCHLORATE/CHLORATE SALT LEVELS WHEN COMPARED TO CONTROL INCUBATIONS AT 0 DEGREE C AND 101.3 KPA IN STANDARD LIQUID MEDIA.THE PROPOSAL IS RELEVANT TO THE C.5 EXOBIOLOGY PROGRAM BASED ON: (1) THE RESEARCH WILL "..SEEK TO DETERMINE THE BIOLOGICAL AND ENVIRONMENTAL FACTORS LEADING TO-THE POTENTIAL DISTRIBUTION OF COMPLEX LIFE IN THE UNIVERSE " AND (2) THE RESEARCH WILL STUDY THE "ESTABLISHMENT OF LIFE UNDER CONDITIONS PREVAILING ON OTHER PLANETARY BODIES..;" NAMELY THE GROWTH OF A PLAUSIBLE METHANOGENIC MICROBIAL COMMUNITY IN ACTIVE RSL FEATURES ON MARS.