Deciphering the electric code of Geobacter sulfurreducens in cocultures with Pseudomonas aeruginosa via SWATH-MS proteomics

Interspecies electron transfer (IET) occurs in many microbial communities, enabling extracellular electron exchange for syntrophic utilization of mixed resources. Various mechanisms of IET have been characterized including direct IET (DIET) and hydrogen IET (HIT) but their evolution throughout syntrophic adaptation has not been investigated through an omics approach. A syntrophic coculture of Geobacter sulfurreducens and Pseudomonas aeruginosa was established and evolved in restricted medium. The medium required cooperative metabolism due to preferential utilization of formate and fumarate by P. aeruginosa and G. sulfurreducens respectively. Pure cultures did not yield significant growth while substantial growth was observed in cocultures. The syntrophy was not reliant on phenazine, since Δphz mutant strain cocultures grew, however appeared to rely on cytochromes as evidenced from the stunted growth G. sulfurreducens ΔomcZ and ΔomcS mutant cocultures. SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) proteomic analysis of initial cocultures revealed upregulation in DIET-associated cytochromes, whereas adapted cocultures revealed upregulation in HybA, a G. sulfurreducens uptake hydrogenase critical to HIT. This suggests DIET plays a critical role in the establishment of syntrophy between G. sulfurreducens and P. aeruginosa but is later consolidated with HIT as the cocultures adapt. This is the first instance to show a temporal distribution of DIET and HIT within the same coculture.