Evolved bacterial formate assimilation is likely potentiated by a rudimentary CO ₂ concentrating mechanism

Formate is a single-carbon compound that is challenging to assimilate, including when assimilation involves a CO ₂ intermediate that can diffuse from the cell. Mutations that overcome such challenges can be identified through adaptive laboratory evolution. We evolved the anoxygenic phototrophic bacterium Rhodopseudomonas palustris to use formate as the sole carbon source. Through gene deletions, we determined that formate is assimilated via oxidation to CO ₂ by formate dehydrogenase followed by CO ₂ fixation by the Calvin cycle. However, this pathway had no clear link to three genes that were commonly mutated in evolved isolates: (i) ribB , a flavin synthesis enzyme, (ii) ppsR2 , a repressor of light-harvesting genes, and (iii) RPA0893, a regulator of unknown function. A RibB mutation was necessary and sufficient for formate assimilation and improved formate oxidation. PpsR2 mutations occurred early, facilitated formate assimilation, and caused elevated pigmentation. Pigment production generates CO ₂ and alkaline conditions that, along with intracellular chromatophore membranes, could represent a rudimentary but important CO ₂ -concentrating mechanism. RPA0983 mutations emerged late and facilitated formate assimilation. RPA0983’s proximity to a CO ₂ -liberating pigment synthesis gene suggests a similar effect as PpsR2 mutations. Our findings reveal unintuitive pathway intersections that could have broad implications for formate and CO ₂ -utilizing organisms.