ABSTRACT
Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope fractionation in cyanobacteria. We found that >2% CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. At elevated CO2, carbon isotope discrimination (εp) is increased by ~8‰, whereas RuBisCO overexpression does not significantly affect isotopic discrimination at all tested CO2 concentrations. Our results show that understanding the environmental factors that impact RuBisCO regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record of organic and inorganic carbon isotope signatures.
IMPORTANCE Carbon isotope biosignatures preserved in the geologic record are interpreted to reflect the long-term evolution of microbial carbon fixation and provide the earliest evidence of life on Earth. RuBisCO enzymes, distinctive and early-evolved catalysts that fix atmospheric CO2, have likely been responsible for the bulk of primary productivity through Earth history. Thus, a comprehensive understanding of the molecular, physiological, environmental, and evolutionary factors that influence the isotopic discrimination of cyanobacteria that utilize RuBisCO is essential for the interpretation of ancient isotopic biosignatures. For example, the vastly different atmospheric CO2 levels that characterized the Precambrian may have influenced the expression and regulation of the ancient RuBisCO protein complex. These observations underscore the need to consider how a broader range of environmental conditions and subcellular processes may have shaped isotopic discrimination over geologic time. In this study, we establish a cyanobacterial metabolic-engineering strategy that can test such hypotheses and offer insights into the biogeochemical record of life.
Competing Interest Statement
The authors have declared no competing interest.
