Activity-targeted metaproteomics uncovers rare syntrophic bacteria central to anaerobic community metabolism

Abstract Syntrophic microbial consortia can contribute significantly to the activity and function of anoxic ecosystems, yet are often too rare to study their in situ physiologies using traditional molecular methods. Here, we combined bioorthogonal non-canonical amino acid tagging (BONCAT), stable isotope probing, and metaproteomics to improve the recovery of proteins from active members and track isotope incorporation in an anaerobic digestion community. Both click chemistry-enabled cell-sorting and direct protein pulldown coupled to metaproteomics improved recovery of isotopically labeled proteins during anaerobic acetate oxidation. Resulting labeled protein expression profiles revealed elevated activity of a rare and so-far uncharacterized syntrophic bacterium belonging to the family Natronincolaceae. BONCAT-based capture of newly translated proteins provided direct molecular evidence for the expression of a previously hypothesized oxidative glycine pathway for syntrophic acetate oxidation by this microorganism, showcasing the potential of targeted metaproteomics to characterize rare and active cells central to community metabolism in natural and engineered ecosystems. Competing Interest Statement With the exception of SJH, the authors declare no competing interests associated with this work. SJH is a co-founder of Koonkie Inc., a bioinformatics consulting company that designs and provides scalable algorithmic and data analytics solutions in the cloud. Koonkie Inc. was not involved in any aspect of this research. Footnotes The bacterial species name has been updated to "Syntrophacetatiphaga salishiae" to reflect the final endorsed naming in the SeqCode database.