Although biofilms are widespread in nature, the ecological roles and compositional diversity of the extracellular polymeric substances (EPS) forming these structures remain poorly understood. Here, we apply a bottom-up genomic approach by investigating the biosynthetic potential for glycan precursors in the genus “Candidatus Accumulibacter”, with a focus on assessing the intra-genus variability. Within a curated set of 61 “Ca. Accumulibacter” MAGs, our analysis revealed a dichotomy in glycan precursors between a conserved core group of 9 nucleotide-sugars and a variable accessory set of 12 nucleotide-sugars, out of 50 nucleotide-sugars tested. The core nucleotide-sugars in “Ca. Accumulibacter” are related to nucleotide-sugars also found to be widely distributed across the tree of life, whereas the accessory set is enriched in rare nucleotide-sugars. The accessory nucleotide-sugars show an irregular distribution across “Ca. Accumulibacter” phylogeny, and divergent evolutionary histories. This highlights the possibility that distinct evolutionary pressures act on different parts of the EPS-formation metabolism, leading to genotypic diversification driven by complex biological phenomena such as horizontal gene transfer that support the observed divergent evolutionary histories.

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 those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. 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.