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.