An interspecies malate–pyruvate shuttle reconciles redox imbalance in an anaerobic microbial community

Abstract

Microbes in ecosystems often develop coordinated metabolic interactions. Therefore, understanding metabolic interdependencies between microbes is critical to deciphering ecosystem function. In this study, we sought to deconstruct metabolic interdependencies in organohalide-respiring consortium ACT-3 containing Dehalobacter restrictus using a combination of metabolic modeling and experimental validation. D. restrictus possesses a complete set of genes for amino acid biosynthesis yet when grown in isolation requires amino acid supplementation. We reconciled this discrepancy using flux balance analysis considering cofactor availability, enzyme promiscuity, and shared protein expression patterns for several D. restrictus strains. Experimentally, 13 C incorporation assays, growth assays, and metabolite analysis of D. restrictus strain PER-K23 cultures were performed to validate the model predictions. The model resolved that the amino acid dependency of D. restrictus resulted from restricted NADPH regeneration and predicted that malate supplementation would replenish intracellular NADPH. Interestingly, we observed unexpected export of pyruvate and glutamate in parallel to malate consumption in strain PER-K23 cultures. Further experimental analysis using the ACT-3 transfer cultures suggested the occurrence of an interspecies malate-pyruvate shuttle reconciling a redox imbalance, reminiscent of the mitochondrial malate shunt pathway in eukaryotic cells. Altogether, this study suggests that redox imbalance and metabolic complementarity are important driving forces for metabolite exchange in anaerobic microbial communities.

A gap-filling algorithm for prediction of metabolic interactions in microbial communities

“…This exchange could indicate that Dehalobacter sp. CF performs CO fixation, which is possible since the species is known to possess the Wood-Ljungdahl pathway [63]. Furthermore, we observed patterns in the amino acid exchanges between the two community members, with arginine, asparagine, isoleucine, lysine, phenylalanine, threonine, and tyrosine transferred from Bacteroidales sp.…”

“…CF50 to consume lactate, while the two models grow anaerobically in the same medium and are allowed to exchange organic acids and amino acids. Based on the experimental study for the coculture [63], after 18 days, the community consumes 0.5 mM of chloroform and 0.75 mM of lactate, while the total cell density is around 6•10 7 cells•mL -1 , and the relative abundance of the Dehalobacter in the community is 66% on average. This information was used in order to calculate the uptake rates of chloroform and lactate by the community as shown in S5 Table . The reaction constraints for the two models are shown in S6 Table and S7 Table . For our simulations, we used 0.1 and 0.01 d -1 as lower bounds for the growth rates of the Dehalobacter sp.…”

“…CF50, respectively. All the constraints applied for the exchange reactions and the growth rates are representing experimental data from the cogrowth of the Dehalobacter and Bacteroidales species [63].…”

“…The study of this community has garnered scientific interest in recent years, as it has the ability to degrade chlorinated environmental pollutants [60][61][62]. The ACT-3 community is mainly composed of bacterial species that belong in the genera Dehalobacter, with relative abundance up to 80%, Bacteroides, and Clostridium [63]. The dechlorinating ability of the ACT-3 community mainly comes from the ability of Dehalobacter species to perform dehalogenation, i.e., anaerobic respiration with the use of chloroform or other chlorinated compounds as electron acceptors [64][65][66][67][68].…”

“…CF and Bacteroidales sp. CF50, in order to simulate experimental results for the cogrowth of the two most abundant species of the ACT-3 community [63] and improve our models.…”