Linking Exoproteome Function and Structure to Anammox Biofilm Development

Extracellular proteins are of paramount importance in the cell–cell interactions of anammox biofilms. However, the inherent aggregation mechanisms of anammox have largely remained elusive. Herein, using a quartz sand extraction protocol and follow-up iTRAQ-based quantitative proteomics, we identified 367 extracellular proteins from initial colonizers, mature biofilm, and detached biofilm. The extracellular proteins were mainly membrane-associated. Most of the recovered proteins (226, 72.5%) originated from the phylum Planctomycetes. In summary, 215 and 190 of the 367 proteins recovered were up- and/or downregulated at least 1.2-fold during the biofilm formation and detachment periods, respectively. These differentially expressed proteins were dominantly involved in metal ion binding, which was regarded as strong evidence for their abilities to enhance ionic bridges in extracellular polymeric substances (EPS). Scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of the biofilms further showed substantial levels of calcium and iron minerals. Critically, representative Sec-dependent secretory proteins affiliated with coccoid Planctomycetes, rod-shaped Proteobacteria, and filamentous Chloroflexi (11, 4, and 2 with differential expression, respectively) were found to have typical and abundant inner β-sheet structures, wherein hydrophobic moieties can promote anammox aggregation. Overall, these findings highlight links between differentially expressed protein functions and morphologic traits of anammox consortia during biofilm development.