Trajectories for the evolution of bacterial CO₂-concentrating mechanisms

Cyanobacteria rely on CO₂-concentrating mechanisms (CCMs) to grow in today's atmosphere (0.04% CO₂). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form-i.e., lacking a single gene-cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was "catalyzed" by historically high CO₂ levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO₂ concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO₂ levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator. To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO₂ and HCO₃^-, as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO₂ induces an HCO₃^- deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.