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Most autotrophs use the Calvin–Benson–Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts (“CandidatusThiobarba”) of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that “Ca. Thiobarba” switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated “Ca. Thiobarba”. Direct stable isotope fingerprinting showed that “Ca. Thiobarba” has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

Most anoxic environments are populated by small (<10?µm) heterotrophic eukaryotes that prey on different microbial community members. How predatory eukaryotes engage in beneficial interactions with other microbes has rarely been investigated so far. Here, we studied an example of such an interaction by cultivating the anerobic marine flagellate, Carpediemonas frisiasp. nov. (supergroup Excavata), with parts of its naturally associated microbiome. This microbiome consisted of so far uncultivated members of the Deltaproteobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia and Nanoarchaeota. Using genome and transcriptome informed metabolic network modeling, we showed that Carpediemonasstimulated prokaryotic growth through the release of predigested biomolecules such as proteins, sugars, organic acids and hydrogen. Transcriptional gene activities suggested niche separation between biopolymer degrading Bacteroidetes, monomer utilizing Firmicutes and Nanoarchaeota and hydrogen oxidizing Deltaproteobacteria. An efficient metabolite exchange between the different community members appeared to be promoted by the formation of multispecies aggregates. Physiological experiments showed that Carpediemonascould also benefit from an association to these aggregates, as it facilitated the removal of inhibiting metabolites and increased the availability of prey bacteria. Taken together, our results provide a framework to understand how predatory microbial eukaryotes engage, across trophic levels, in beneficial interactions with specific prokaryotic populations.