MOTIVATION: A CHALLENGE FOR EXOBIOLOGY IS TO EXPLAIN THE EVOLUTION OF PHOTOSYNTHESIS A COMPLEX PATHWAY REQUIRING SEVERAL COOPERATIVE ENZYMES AND THE SYNTHESIS AND USE OF MULTIPLE COFACTORS. ANOXYGENIC PHOTOSYNTHESIS IN PARTICULAR IS IMPORTANT FOR THE EARLY ANOXIC EARTH AND FOR EXOPLANETS WHERE ANOXIA MIGHT BE QUITE COMMON. IF THE EVOLUTION OF PHOTOSYNTHESIS VIA A FEW 'RELATIVELY EASY' STEPS IS POSSIBLE WE MIGHT EXPECT TO FIND MANY WORLDS WITH PHOTOSYNTHETIC LIFE WHETHER OR NOT OXYGENIC PHOTOSYNTHESIS EVER EMERGED. OTHERWISE WE MIGHT EXPECT PHOTOSYNTHESIS TO BE RARE IN THE UNIVERSE. WE PROPOSE THAT A PLAUSIBLE EVOLUTIONARY INTERMEDIATE IN THE DEVELOPMENT OF (ANOXYGENIC) PHOTOSYNTHESIS WAS A CHEMOLITHOTROPHIC BACTERIUM THAT EXTRACTED ENERGY FROM ENVIRONMENTAL REDOX GRADIENTS. ONE WAY TO EXTRACT MORE ENERGY FROM A REDOX GRADIENT IS TO INSERT A PHOTOSYNTHETIC REACTION CENTER (RC) INTO THE ELECTRON TRANSPORT PATHWAY. THIS GIVES A CHEMOLITHOTROPH A COMPETITIVE ADVANTAGE ALLOWING IT TO USE LESS-REDUCING DONORS AND/OR LESS-OXIDIZING ACCEPTORS. A SHALLOW GRADIENT OR EVEN AN UPHILL ELECTRON TRANSFER COULD BE PROFITABLE TO AN ORGANISM USING LIGHT TO GENERATE A STEEPER GRADIENT THUS PUMPING MORE PROTONS PER ELECTRON TRANSPORTED AND MAKING MORE ATP. IN THIS VIEW THE REACTION CENTER ALLOWS THE DEVELOPMENT OF LIGHT-ASSISTED LITHOTROPHY I.E. PHOTO/ CHEMOLITHOTROPHY. OBJECTIVES: THERE ARE NO KNOWN EXAMPLES OF THE PHOTO/CHEMOLITHOTROPH WE INVOKE IN THIS EVOLUTIONARY TRAJECTORY. WE PROPOSE TO TEST THIS IDEA BY CREATING SUCH ORGANISMS. WE WILL USE THE PRIMITIVE PHOTOTROPH HELIOBACTERIUM MODESTICALDUM AS OUR STARTING POINT BECAUSE IT IS EXPERIMENTALLY MUCH EASIER TO TRANSFER A FEW GENES REQUIRED TO CONFER LITHOTROPHY RATHER THAN THE MANY GENES REQUIRED FOR PHOTOTROPHY. WE HAVE DEVELOPED GENETIC TOOLS TO EDIT THE H. MODESTICALDUM CHROMOSOME USING ITS ENDOGENOUS DNA MODIFICATION SYSTEMS; THIS ALLOWS US TO REMOVE AND ADD GENES AT WILL. WE PROPOSE TO BREAK THE EXISTING CYCLIC ELECTRON FLOW PATHWAY BY REMOVING A SPECIFIC ENZYME (NADH DEHYDROGENASE) AND ADDING A SULFIDE:QUINONE OXIDOREDUCTASE (SQR) TO ALLOW IT OXIDIZE SULFIDE TO SULFUR. THE ORGANISM CAN ALREADY REDUCE PROTONS TO MOLECULAR HYDROGEN (H2). THEREFORE IT SHOULD BE ABLE TO OXIDIZE (EXTERNAL) SULFIDE AND REDUCE (INTERNAL) PROTONS AIDED BY LIGHT AND IN THE PROCESS CREATE A PROTON ELECTROCHEMICAL GRADIENT FOR ATP SYNTHESIS THUS EXTRACTING ENERGY FROM A REDOX GRADIENT THAT IS ENERGETICALLY UNFAVORABLE. WE WILL TEST THE GROWTH OF THE ENGINEERED ORGANISMS UNDER DIFFERENT CONDITIONS AND QUANTIFY THEIR ABILITY TO (1) USE SULFIDE (S-2) AS ELECTRON DONOR (2) REDUCE PROTONS TO H2 (3) EXTRACT ENERGY IN THE PROCESS AND (4) FIX CO2. WE WILL ALSO TEST THE IDEA THAT AUTOTROPHY CAN BE CONFERRED BY ADDING ATP-CITRATE LYASE WHICH IS THE ONLY ENZYME MISSING FROM THE REVERSE TCA CYCLE IN H. MODESTICALDUM. WE CAN MAKE SEVERAL COMBINATIONS OF THESE GENETIC MODIFICATIONS IN ORDER TO CONSTRUCT MULTIPLE POSSIBLE EVOLUTIONARY INTERMEDIATES. LASTLY WE WILL ASSESS THE ISOTOPE SIGNATURES ASSOCIATED WITH THE NEW SULFIDE OXIDATION PROTON REDUCTION AND CO2 FIXATION PATHWAYS AND COMPARE THEM WITH KNOWN METABOLIC FRACTIONATIONS AND WITH THE GEOLOGIC RECORDS. SIGNIFICANCE: THIS WORK IS RELEVANT FOR NASA EXOBIOLOGY BECAUSE IT DIRECTLY INVESTIGATES TWO RESEARCH TOPICS IN THE 2015 ASTROBIOLOGY STRATEGY: EARLY LIFE AND INCREASING COMPLEXITY AND CO-EVOLUTION OF LIFE AND THE PHYSICAL ENVIRONMENT. SPECIFICALLY WE WILL EXPERIMENTALLY EXPLORE THE ORIGIN AND EVOLUTION OF A KEY METABOLIC STRATEGY ANOXYGENIC PHOTOTROPHY IN THE CONTEXT OF ENVIRONMENTAL REDOX GRADIENTS. OUR RESULTS WILL PROVIDE INSIGHT INTO THE POTENTIAL BIOSIGNATURES OF THE METABOLIC STRATEGIES WE EXPLORE IN THE ENGINEERED H. MODESTICALDUM STRAINS. THE RESULTS WILL HAVE IMPLICATIONS FOR UNDERSTANDING THE EVOLUTION OF LIFE ON THE EARLY EARTH AND FOR THE DIVERSITY OF BIOSIGNATURES THAT MIGHT RESULT FROM NOVEL METABOLISMS.