Evolved formate assimilation in phototrophic Rhodopseudomonas palustris indicates the need for a CO₂-concentrating...

Formate is a single-carbon compound that is challenging to assimilate, including when assimilation involves a CO₂ 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 CO₂ by formate dehydrogenase, followed by CO₂ fixation by the Calvin cycle. However, this pathway had no clear link to three genes that were commonly mutated in evolved isolates: (1) ppsR2, a repressor of light-harvesting genes; (2) ribB, a flavin synthesis enzyme; and (3) RPA0853, a regulator of unknown function. PpsR2 mutations occurred early and facilitated formate assimilation but were insufficient for formate assimilation on their own. Pigment production generates CO₂ and alkaline conditions that, along with intracellular chromatophore membranes, could represent a rudimentary but important CO₂-concentrating mechanism. A RibB mutation was necessary and sufficient for formate assimilation and improved formate oxidation to CO₂. RPA0853 mutations emerged late, in some cases, and facilitated formate assimilation. The mutant phenotypes had a common theme of CO₂ production and retention, indicating that maintaining CO₂ availability is an obstacle for formate assimilation via the Calvin cycle. The unintuitive pathway intersections could have broad implications for understanding formate and CO₂-utilizing organisms.