TWO EXOBIOLOGY PROGRAM GOALS AS DESCRIBED IN THE ROSES 2017 NASA RESEARCH ANNOUNCEMENT ARE TO UNDERSTAND 'EARLY EVOLUTION OF LIFE AND THE BIOSPHERE' AND 'BIOSIGNATURES AND LIFE ELSEWHERE'. IN KEEPING WITH THESE GOALS THIS PROPOSAL ADDRESSES TWO CENTRAL RESEARCH QUESTIONS: 1) HOW CAN A HYPERTHERMOPHILIC CRENARCHAEON GAIN ENERGY ON ITS CYTOPLASMIC MEMBRANE BY RESPIRING AN INSOLUBLE IRON OXIDE MINERAL AND 2) DOES THIS ORGANISM PRODUCE CYTOCHROMES OR MINERALS DURING IRON REDUCTION THAT PROVIDE A UNIQUE SPECTRAL BIOSIGNATURE? THE GOALS OF THE PROJECT ARE TO IMPROVE OUR UNDERSTANDING OF THE ORIGIN AND CORE COMPONENTS OF MICROBIAL IRON REDUCTION MODEL THE PHYSIOLOGICAL MECHANISM OF ELECTRON TRANSFER FROM CELLS TO IRON OXIDE MINERALS EXPAND THE SUITE OF POSSIBLE SPECTRAL BIOSIGNATURES IMPROVE AVAILABLE DETECTION USING SPACECRAFT-RELEVANT INSTRUMENTATION AND INCREASE THE COMPUTATIONAL ROBUSTNESS OF BIOSIGNATURE INTERPRETATION. THIS PROJECT USES THE HYPERTHERMOPHILIC H2-OXIDIZING CRENARCHAEON AND IRON REDUCER PYRODICTIUM DELANEYI AS ITS MODEL ORGANISM. P. DELANEYI WAS ISOLATED FROM A DEEP-SEA HYDROTHERMAL VENT 'BLACK SMOKER' CHIMNEY. THE ORGANISM AND ENVIRONMENT ARE REPRESENTATIVE OF EARLY LIFE ON EARTH AND MODELS FOR POTENTIAL LIFE BEYOND EARTH. THE P. DELANEYI GENOME ENCODES FOUR NOVEL RESPIRATORY COMPLEXES THAT ARE ABSENT IN OTHER ARCHAEAL FAMILIES. IT LACKS THE GENES FOR OTHER KNOWN ARCHAEAL IRON REDUCTION MECHANISMS BUT HAS 15 GENES FOR NOVEL C-TYPE CYTOCHROME PROTEINS. MULTIHEME C-TYPE CYTOCHROMES ARE GENERALLY REQUIRED FOR MICROBIAL IRON REDUCTION. THE PROJECT HAS THREE THEMES: 1. PHYSIOLOGICAL MECHANISM OF IRON REDUCTION. WE WILL IDENTIFY THE RESPIRATORY COMPLEX(ES) USED BY P. DELANEYI TO REDUCE THE IRON OXIDE MINERAL FERRIHYDRITE TO MAGNETITE. 2. SPECTRAL DIFFERENTIATION OF HYPERTHERMOPHILES. WE WILL CHARACTERIZE THE SPECTRAL PROPERTIES OF P. DELANEYI BIOMASS GROWN ON FERRIHYDRITE RELATIVE TO ITS GROWTH ON NITRATE AND TO OTHER HYPERTHERMOPHILES. 3. SPECTRAL DIFFERENTIATION OF MINERALS. WE WILL IDENTIFY THE FE(II) MINERALS FORMED BY P. DELANEYI DURING IRON RESPIRATION RELATIVE TO THOSE FORMED BY OTHER IRON-REDUCING HYPERTHERMOPHILES AND FORTUITOUSLY BY NON-IRON-REDUCING HYPERTHERMOPHILES. P. DELANEYI WILL BE GROWN ON FERRIHYDRITE AND NITRATE. DIFFERENTIAL GENE AND PROTEIN EXPRESSION WILL IDENTIFY IRON-RESPIRATION SPECIFIC PROTEIN COMPLEXES USING RNA-SEQ PROTEOMICS CYTOCHROME C HEME STAINING AND PEPTIDE MASS FINGERPRINTING. SOME HYPERTHERMOPHILES POSSESS DIFFERENT TYPES AND PROPORTIONS OF LIGHT-ABSORBING PIGMENTS (E.G. CYTOCHROMES COENZYME F420) THAT MAY CHANGE WITH METABOLISM. THEREFORE VISIBLE-TO-NEAR INFRARED AND MID-INFRARED REFLECTANCE AND RAMAN SPECTROSCOPIES WILL BE USED TO DIFFERENTIATE P. DELANEYI BIOMASS GROWN ON IRON AND NITRATE AS WELL AS THE BIOMASSES OF OTHER HYPERTHERMOPHILES (E.G. PYROBACULUM THERMOCOCCUS METHANOCALDOCOCCUS). THESE SPECTROSCOPIES WILL LIKEWISE BE USED TO DIFFERENTIATE THE REDUCED IRON MINERAL PRODUCTS OF P. DELANEYI GROWN ON FERRIHYDRITE WITH THOSE OF OTHER IRON-REDUCING HYPERTHERMOPHILES AND WITH NON-IRON REDUCING HYPERTHERMOPHILES THAT FORTUITOUSLY REDUCE FERRIHYDRITE WHEN GROWN IN ITS PRESENCE. THIS PROJECT FULFILLS MANY OF THE EXOBIOLOGY PROGRAM'S STATED GOALS NAMELY TO "1) UNDERSTAND THE PHYLOGENY AND PHYSIOLOGY OF MICROORGANISMS INCLUDING EXTREMOPHILES WHOSE CHARACTERISTICS MAY REFLECT THE NATURE OF PRIMITIVE ENVIRONMENTS 2) DETERMINE THE ORIGINAL NATURE OF BIOLOGICAL ENERGY TRANSDUCTION MEMBRANE FUNCTION AND INFORMATION PROCESSING TO TEST HYPOTHESES REGARDING THE ORIGINAL NATURE OF KEY BIOLOGICAL PROCESSES AND 3) INVESTIGATE THE DEVELOPMENT OF KEY BIOLOGICAL PROCESSES AND THEIR ENVIRONMENTAL IMPACT." IT ALSO SERVES THE GOALS OF THE 2015 NASA ASTROBIOLOGY STRATEGY DOCUMENT INCLUDING CO-EVOLUTION OF LIFE AND THE PHYSICAL ENVIRONMENT; AND IDENTIFYING EXPLORING AND CHARACTERIZING ENVIRONMENTS FOR HABITABILITY AND BIOSIGNATURES.