Evolution of Carbon Isotope Fractionation in Cyanobacteria

The history of Earths carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass ({varepsilon}p) and enzymatic ({varepsilon}Rubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to >>1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger {varepsilon}p values than WT, despite having a much smaller {varepsilon}Rubisco (17.23 {+/-} 0.61{per thousand} vs. 25.18 {+/-} 0.31{per thousand} respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result--ANC {varepsilon}p exceeded ANC {varepsilon}Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO2 concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to {varepsilon}p, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2. Significance StatementEarth scientists rely on chemical fossils like the carbon isotope record to derive ancient atmospheric CO2 concentrations, but interpretation of this record is calibrated using modern organisms. We tested this assumption by measuring the carbon isotope fractionation of a reconstructed ancestral rubisco enzyme (>1 billion years old) in vivo and in vitro. Our results contradicted prevailing models of carbon flow in Cyanobacteria, but our data could be rationalized if light-driven uptake of CO2 is taken into account. Our study showed that the carbon isotope record tracks both the evolution of photosynthesis physiology as well as changes in atmospheric CO2, highlighting the value of considering both evolution and physiology for comparative biological approaches to understanding Earths history.