Metaproteomic profiling of the secretome of a granule-forming Ca . Accumulibacter enrichment

ABSTRACT Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture’s supernatant successfully identified over 50% of the protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. However, while most filamentous proteins originated from Ca . Accumulibacter, among others cell surface proteins did not. This suggests that not only a range of different proteins, but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca . Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general. SIGNIFICANCE Biofilm-forming microbial communities are widespread and pose both challenges and opportunities in various settings in life. Structure-providing, extracellular proteins likely play a crucial role in the formation of the biofilm matrix, but these proteins are challenging to characterise due to the dynamic and complex nature of these communities. We used two advanced metaproteomic approaches to enrich for the extracellular proteins in a granule-forming Candidatus Accumulibacter enrichment culture as a proxy for granule-forming communities present in wastewater treatment plants. The extracellular proteins were additionally classified using structure and sequence-based annotation tools, which identified multiple different protein categories that potentially aid in granule formation, but also may provide structure to the biofilm matrix. Interestingly, although the granules were highly enriched for Ca . Accumulibacter, several structure-providing protein categories originated from other organisms. The obtained metaproteomic data contribute to the understanding of proteins and processes that are potentially involved in granule formation. This could further help to optimise processes involving granular biofilms and identify candidates for the recovery of novel biopolymers for biotechnological applications.