Reductive TCA cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin cycle mutant

Purple nonsulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Excess reduced electron carriers must be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize excess reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin cycle mutant maintains electron balance under such conditions. We detected the reversal of some TCA cycle enzymes, which carried reductive flux from malate or fumarate to -ketoglutarate. This pathway and the reductive synthesis of amino acids derived from -ketoglutarate are likely important for electron balance, as supplementing the growth medium with -ketoglutarate-derived amino acids prevented Rs. rubrum Calvin cycle mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.