Chemoautotrophy–which is the anchor that embeds the biosphere within geochemistry– is grounded on carbon-fixation, the transformation of C1 compounds into small organic molecules and hence into the large array of organic compounds that constitute metabolism. Carbon monoxide (CO) is the most abundant C source in the cosmic gas phase, and follows after hydrogen as the most common gas in the universe. This proposal seeks to model the evolution of metabolism for carbon fixation into early life forms and we consider utilization of CO an excellent candidate for the carbon and energy source in the first life forms. Anaerobic chemoautotrophy, the fixation of C1 compounds in the absence of light and oxygen, is widespread among deeply branching thermophilic bacteria and archaea, through five recognized pathways. In comparing C1 acquisition systems in extant life, the reductive acetyl-CoA pathway (also known as the Wood-Ljungdahl pathway) allows for growth on various carbon sources (e.g. CO, CO2, formaldehyde) and has the lowest energy requirement and minimal need for de novo protein synthesis of the characterized carbon acquisition pathways. The capacity to fix carbon monoxide and the extreme oxygen sensitivity of its key enzymes are the grounds for our hypothesis that the Wood-Ljungdahl (WL) pathway might be the original carbon fixation pathway. Despite the key role of this pathway in the global carbon cycle, knowledge of its regulation and evolution under low-energy conditions is limited. Therefore we propose to investigate the evolution of the carbon fixation strategies in the two thermophilic chemolithoautotrophs, Carboxydothermus hydrogenoformans and Thermovibrio ammonificans and their adaptations to low energy conditions. These strictly anaerobic bacteria are able to grow in pure culture on the cosmically abundant gases CO, H2 and CO2, and each could form autonomous one-species ecosystems in minimally complex growth conditions. Thus we refer to them as sentinel organisms. Both strains encode the WL pathway, however, Thermovibrio ammonificans also encodes an alternative CO2 fixation pathway, reductive tricarboxylic acid cycle (rTCA), making it an excellent model system to investigate the interplay of parallel carbon fixation strategies under energy 'famine' or 'feast' conditions. The prototype carboxydotroph Carboxydothermus hydrogenoformans can grow by utilizing CO across the microbial "feast" to "famine" range for CO, from 1.3 atm of CO down to below 2 ppmv, the limit of detection. Our strategy includes the recombinant expression of genes and gene transcripts encoding for key enzymes in the dark CO fixation pathways of the model organisms. We will exert both positive and negative selection on the WL pathway under C-limited growth conditions, to determine regulatory mechanisms and whether genomic adaptation occurs in response to selection for increased fitness in carbon limited conditions The proposed research addresses Exobiology program goals (i) determine when and in what setting life first appeared and the characteristics of the first successful living organisms; and (ii) understand the phylogeny and physiology of microorganisms, including extremophiles, whose characteristics may reflect the nature of primitive environments.